Ionized magnesium2+ concentrations in biological samples

ABSTRACT

The present invention provides a method for preparing and storing biological samples prior to measuring ionized Mg 2+  concentrations in order to obtain accurate and reproducible readings. A novel method for accurately reading ionized magnesium ion concentrations in biological samples is provided. A method for monitoring ionized Mg 2+  concentrations and ionized Ca 2+  :Mg 2+  ratios in a patient, useful in diagnosing and prognosing disease states including cardiopulmonary bypass surgery, hypertension, abnormal pregnancy, head trauma, and fetal abnormalities is provided as well as a composition and a method of treating a patient with ionized Mg 2+  or ionized Ca 2+  and Mg 2+ .

This is a division of co-pending application Ser. No. 07/864,646 filedApr. 7, 1992 which is a continuation-in-part of copending applicationSer. No. 07/681,940 filed Apr. 8, 1991.

BACKGROUND OF THE INVENTION

Magnesium (Mg) is the second most abundant cation in the body [Altura,B. M. et al., Drugs 28 (Suppl. I): 120-142, 1984]. It is cofactor formore than 325 cellular enzymes involved in cellular energy productionand storage, protein synthesis, DNA and RNA synthesis, cell growth andreproduction, adenylate cyclase synthesis, maintenance of cellularelectrolyte composition, and stabilization of mitochondrial membranes[Altura, B. M. et al, Drugs 28 (Suppl. I): 120-142, 1984; Wacker, W. E.C. Magnesium and Man, Harvard Univ. Press, Cambridge, 1980]. As aconsequence of these biochemical activities, Mg plays a pivotal role incontrol of neuronal activity, cardiac excitability, neuromusculartransmission, muscular contraction, and vasomotor tone [Altura, B. M. etal., Drugs 28 (Suppl. I): 120-142, 1984; Wacker, W. E. C. Magnesium andMan, Harvard Univ. Press, Cambridge, 1980; Altura, B. M. et al., in:Metal Ions in Biological Systems, ed. by H. Sigel and A. Sigel, Vol 26:Compendium on Magnesium and Its Role in Biology, Nutrition, andPhysiology, pp 359-416, Marcel Dekker, Inc. New York, 1990].

Most clinical data of Mg determinations are derived from blood levels oftotal Mg (Wacker, W. E. C. Magnesium and Man, 1980; Elin, R. J. Clin.Chem. 33:1965-1970, 1987). Total serum Mg concentrations reflectprotein-bound (30-40%), chelated (7-12%), and free or ionized Mg (Mg²⁺)(60-70%) fractions. The exact proportion of these fractions has beenextremely difficult to determine precisely, and, moreover, there is noway to rapidly make such determinations. Precise information about Mgactivity is pivotal to our understanding of Mg metabolism. The free orionized form (Mg²⁺) is the active form of the mineral (Wacker, W. E. C.Magnesium and Man, 1980; Elin, R. J. Clin. Chem. 33:1965-1970, 1987;Ryan, M. F. Ann. Clin. Biochem. 28:19-26, 1991). Alterations incirculating protein levels (primarily albumin), which are seen innumerous pathophysiologic states, will alter the interpretation of Mgstatus (very similar to calcium) (Elin, R. J. Clin. Chem. 33: 1965-1970,1987).

Although numerous methods are available clinically, to determine totalMg in serum, plasma, urine, cerebral spinal fluid and other body fluids(e.g., atomic absorption spectrophotometry, atomic emissionspectrophotometry, colorimetry, fluorometry, compleximetry andchromatograph for quantifying total Mg), none of these can determineionized or free Mg²⁺ (Elin, R. J. Clin. Chem. 33:1965-1970, 1987; Wills,M. R. et al. Magnesium 5:317-327, 1986).

Until the present invention, the only method for assessing free Mg²⁺ inbiological samples was an ultrafiltration procedure (Wacker, W. E. C.Magnesium and Man, 1980; Elin, R. J. Clin. Chem. 33:1965-1970, 1987;Wills, M. R. et al. Magnesium 5:317-327, 1986; Aikawa, J. K. Magnesium:Its Biologic Significance, CRC Press, Boca Raton, 1981). While thisprocedure is capable of measuring free Mg²⁺, it is fraught with amultiplicity of problems (need to control pH, need to control filtercomposition, time-consuming, inability to access whole blood Mg²⁺, needfor centrifugation of blood). In addition, and most important, theseclassical methods, which primarily depend upon modifications of theprocedure outlined by Watchorn, E. et al. (Biochem. J. 26:54, 1932),Toribara et al. (J. Clin. Invest. 36:738, 1957) and Walser, M. (J. Clin.Invest. 40:723-730, 1961) result in ionized Mg²⁺ values on normalsubjects which are significantly different from those obtained by thepresent method as assessed using an ion selective electrode (ISE). Usingultracentrifugation methods combined with ultrafiltration methods toassess free Mg²⁺, the percentages of ultrafilterable Mg reported byprevious workers (around 70% ) (Cummings, N. A. et al. Anal. Biochem22:108-116, 1968; Nielson, S. P. Scand. J. Clin. Lab. Invest.23:219-225, 1960) are much higher than the values using the presentmethod. Even more recent measurements, using ultrafiltration and amicropartition filtration system has yielded a much wider range ofvalues for ultrafilterable Mg from normal human subjects than those ofthe present method (D'Costa, M. et al. Clin. Chem. 29:519, 1983; Zaloga,G. P. et al. Crit. Care Med. 15:813-816, 1987). Some of these pitfallspreclude determination of Mg²⁺ in various body fluids. Moreover,determinations can not be done on less than 1.0 ml of blood.

The physiologic or pathophysiologic effects of mild to severe (orgraded) decreases or increases in extracellular free Mg²⁺ in wholeblood, serum or plasma has not been possible to discern in humansubjects or animals either rapidly (e.g., within 1-2 min) or repeatedly(multiple samples over a few minutes-hours). Since Mg is frequently usedin normomagnesemic patients for its antiarrhythmic, vasomotor andneuronal actions [Altura, B. M. et al. Drugs 28(Suppl. I): 120-142,1984; Wacker, W. E. C. Magnesium and Man, 1980; Altura, B. M. et.al. In:Metal Ions in Biological Systems, 1990; Iseri C. T. et al. West J. Med.138:823-828, 1983; Ebel, H. et al. J. Clin. Chem. Clin. Biochem.21:249-265, 1983], it is vital to be able to assess the exactextracellular level of ionized Mg²⁺ at any one instant. Although thereis a dire need to carefully monitor extracellular Mg²⁺ in hypomagnesemicpatients or patients linked to Mg deficiency states such ascardiovascular insufficiency, cardiac arrhythmias, coronary arteryspasm, those at risk for sudden death, renal disorders, respiratorymuscle weakness, pre-eclampsia, eclampsia, migraine, hypertension,premenstrual syndrome, letany, seizures. tremor, apathy, depression,hypokalemia and hypocalcemia, there is at present no way to do thiseither precisely or rapidly [Altura, B. M. et al. Drugs 28(Suppl. I):120-142, 1984; Wacker, W. E. C. Magnesium and Man, 1980; Altura, B. M.et.al. In: Metal Ions in Biological Systems, 1990; Iseri, C. T. West J.Med. 138:823-828, 1983; Ebel, H. et al. J. Clin. Chem. Clin. Biochem.21:249-265, 1983; Altura, B. M. et al. Magnesium 4:226-244, 1985;Zaloga, G. P. Chest 56:257-258, 1989; Sjogren, A. J. Intern. Med.226:213-222, 1989; Zaloga, G. P. et al. In: Problems in Critical Care,ed. G. P. Zaloga Vol 4:425-436, J. B. Lippincott Co., Philadelphia,1990; Resnick, L. M. et al. Proc. Nat. Acad. Sci. USA 81:6511-6515,1984; Rudnick, M. et al. APMIS 98:1123-1127, 1990].

In 1980, it was suggested on the basis of in-vitro experiments thatdrops in ionized serum Mg²⁺ would produce coronary vasospasm,arrhythmias and sudden death (Turlapaty and Altura, Science 208:198-200,1980). Although clinical observations from other workers in theintervening years have suggested this might be a "real" possibility, upuntil the present invention, no evidence could be gathered due to theunavailability of a method for accurate and rapid assessment of bloodionized Mg²⁺ (Altura, B. M. et al. In: Metal Ions in Biological Systems,Vol 26, 1990; Ebel, H. et al. J. Clin. Chem. Clin. Biochem. 21:249-265,1983; Altura. B. M. et al. Magnesium 4:226-244, 1985; Sjogren, A. et al.J. Intern. Med. 226:213-222, 1989; Zaloga, G. P. et al. In: Problems InCritical Care Vol 4, 1990).

Over the past 10 years, it has been determined that reductions inionized Mg²⁺, experimentally in animals and isolated cerebral bloodvessels, can induce intense vasospasm and rupture of blood vessels inthe brain (Altura, B. M. et al. In: Metal Ions in Biological Systems Vol26, 1990; Altura, B. T. et al. Neuroscience Letters 20:323-327, 1980;Altura, B. T. et al. Magnesium 1:277-291, 1982; Altura, B. T. et al.Magnesium 3:195-211, 1984; Altura, B. M. et al. Am. J. Emerg. Med.1:180-193, 1983; Huang, Q-F., et al. FASEB J. 3:A845, 1989). On thebasis of such experimental findings, it has been hypothesized that headtrauma would be associated with deficits in serum, plasma and wholeblood ionized Mg²⁺ (Altura, B. T. et al. Magnesium 1:277-291, 1982;Altura, B. T. et al. Magnesium 3:195-211, 1984; Altura, B. M. et al. Am.J. Emerg. Med. 1:180-193, 1983). The present inventions has allowedthese studies to be undertaken for the first time.

In the 1970's and 1980's, on the basis of numerous animal experiments,it was reported that deficits in ionized Mg²⁺ would result in maintainedperipheral vasospasm, constriction of small blood vessels in numerousorgan regions and as a consequence development of high blood pressure orhypertension (Altura, B. M. et al. Drugs 28 (Suppl. I): 120-142, 1984;Altura, B. M. et al. In: Metal Ions in Biological Systems Vol 26, 1990;Altura, B. M. et al. Magnesium 4:226-244, 1985; Sjogren, A. et al. J.Intern. Med. 226:213-222, 1989; Turlapaty, P. D. M. V. et al. Science208:198-200, 1980; Altura, B. M. et al. Federation Proc. 40:2672-2679,1981; Altura, B. M. et al., Science 221:376-378, 1983; Altura, B. M. etal. Science 223:1315-1317, 1984). Until the development of the presentinvention, this hypothesis was not testable because of a lack of propermethodology for processing samples and measuring ionized Mg²⁺.

Accelerated atherosclerotic heart disease is a leading cause of death inthe long-term (>10 year) renal transplant recipient. Hypertension andhyperlipidemia are common in this population and may be secondary tocyclosporine use. Cyclosporine has been associated with a renal tubulartotal magnesium (TMg) leak, as evidence by low serum total magnesiumvalues and increased urinary excretion. Hypomagnesemia has beenimplicated as a factor in modulation of blood lipid levels, alterationof vascular tone and cyclosporine toxicity. Until the present invention,accurate measurements of biologically active ionized magnesium orionized Ca²⁺ /ionized Mg²⁺ ratios were not possible. Therefore, untilthe present invention, it was not possible to determine the ratio ofionized calcium and ionized magnesium in hypercholesterolemia andcyclosporine toxicity in renal transplant recipients.

In 1981-1983, studies on isolated blood vessels from animals andpregnant women, suggested that reduction in dietary intake of Mg orinability to metabolize Mg properly could result in reduction in ionizedMg²⁺ and thus in umbilical and placenta/vasospasm, possibly reducingoxygen and nutrients to the growing fetus (Altura, B. M. et al.Federation Proc. 40:2672-2679, 1981; Altura, B. M. et al., Science221:376-378, 1983). The end result could be, in large measure,responsible for fetal growth retardation, pre-eclampsia, hypertensionand convulsions, particularly in pregnant indigent women (Rudnick, M. etal. APMIS 98:1123-1127, 1990; Altura, B. M. Science 221:376-378, 1983).Mg has been recommended as early as 1925 in this country for treatmentand prevention of pregnancy-induced pre-eclampsia, hypertension andconvulsions, but a method for accurately monitoring ionized Mg²⁺ rapidlyand repeatedly throughout pregnancy was not available until developmentof the present invention.

A novel method to draw, handle, process and store biological samples foraccurate, rapid and reproducible levels of ionized or free Mg²⁺ wasdeveloped. The method of collecting and processing samples has utilityin preparing biological samples for measurement of ionized Mg²⁺concentrations using a novel selective ion electrode with neutral careerbased membrane. Using the methods of the present invention, an accuratenormal range for ionized Mg²⁺ in whole blood, plasma and serum has beendetermined for the first time. It is now possible to diagnose, prognosesand treat various disease states by the method of the present invention,by monitoring fluctuations in ionized Mg²⁺ concentrations.

SUMMARY OF THE INVENTION

The present invention relates to a method for preparing biologicalsamples, including collection and storage conditions, prior to testingfor ionized Mg²⁺ concentrations under conditions which minimize orprevent exchange of gases between the biological sample and atmosphericair and in which air and O₂ is substantially excluded from thebiological sample, preferably under anaerobic conditions, prior tomeasuring ionized Mg²⁺.

Another aspect of the invention is a method for determining ionized Mg²⁺concentrations in a biological sample, collected and maintained underanaerobic conditions or conditions which minimize or prevent exchange ofgases between the biological sample and atmospheric air and in which O₂is substantially excluded from the biological sample, the Mg²⁺concentration being measured using an ion selective electrode with aneutral carrier based membrane.

Another aspect of the invention is a method for determining ionized Ca²⁺:Mg²⁺ ratios in a biological sample, collected and maintained underanaerobic conditions or conditions which minimize or prevent exchange ofgases between the biological sample and atmospheric air and in which O₂is substantially excluded from the biological sample, the Ca²⁺ and Mg²⁺concentrations being measured using ion selective electrodes with aneutral carrier based membrane.

An additional aspect of the invention is a method for diagnosing orprognosing disease states such as cardiac diseases, hypertension,idiopathic intracranial hypertension, diabetes, lung diseases, abnormalpregnancy, pre-eclampsia, eclampsia, head trauma, fetal growthretardation, and the like, in a patient using a method of determiningionized Mg²⁺ concentrations or ionized Ca²⁺ :Mg²⁺ ratios.

A further aspect of the invention is a method of maintaining normalionized Mg²⁺ concentrations in a patient in need of such maintenancecomprising administration Of Mg²⁺ in the form of a pharmaceuticalcomposition or dietary supplement. Another aspect of the invention is amethod of maintaining normal ionized Ca²⁺ /ionized Mg²⁺ molar ratios inan individual comprising administration of an effective amount of Ca²⁺and Mg²⁺ in the form of a pharmaceutical composition or dietarysupplement.

Another aspect of the invention is a pharmaceutical composition ordietary supplement for preventing or treating magnesium deficiencies,the composition comprising as the active ingredient(s) a concentrationof bioavailable magnesium or bioavailable calcium and magnesium, whereinthe concentration(s) provides a normal physiological molar ratio ofionized Ca²⁺ /Mg²⁺ in the body fluids, such body fluids comprising wholeblood, serum, plasma, cerebral spinal fluid or the like.

BRIEF DESCRIPTION OF DRAWINGS

These and other objects, features, and many of the attendant advantagesof the invention will be better understood upon a reading of thefollowing detailed description when considered in connection with theaccompanying drawings wherein:

FIG. 1 shows the plasma ionized Mg²⁺ concentrations from the whole bloodwhich was spun to pack the formed elements such as erythrocytes, andstored anaerobically for 4 hours at room temperature. It shows thatwhole blood can be stored for at least 4 hours if stored anaerobicallywithout affecting the ionized Mg²⁺ values as compared to an ionized Mg²⁺concentration for fresh plasma.

FIG. 2 shows that freeze-thawing samples has virtually no effect on theionized Mg²⁺ values for samples stored under anaerobic conditions. Incontrast, ionized Ca²⁺ values decline with repeated freeze-thawing ofthe sample.

FIG. 3 aqueous Mg²⁺ vs. aqueous TMg. Correlation of ionized magnesium(Mg²⁺) values by ion selective electrode (ISE) with total magnesium(TMg) values by atomic absorption spectroscopy taken on unbufferedaqueous solutions of MgCl₂.

FIG. 4 Mg²⁺ and Ca²⁺ changes in aqueous buffered solutions with changesin pH as determined by an ISE.

FIG. 5 shows the correlation between serum Mg²⁺ values as measured onultrafiltrate Mg²⁺ by atomic absorption vs. ionized Mg²⁺ valuesdetermined by an ISE. There was a correlation of R=0.88.

FIG. 6 Mg²⁺ (IMg²⁺) and TMg in three patients undergoing cardiacsurgery. Img²⁺ and TMg were followed in three cardiac patientsperioperatively. For each patient, two samples were obtained prior tothe addition of cardioplegia (circled). The first samples obtainedfollowing the addition of cardioplegia (which contained magnesium salt)demonstrated large increases in both the Mg²⁺ and the TMg values (arrowsleft to right). As surgery progressed, both values decreased in eachpatient as indicated by the arrows drawn right to left. The xy line isthat seen in FIG. 3: y=0.71 x+0.01. The average IMg²⁺ /TMg ratiofollowed by the range of ratios for each patient were as follows: filledcircles, 0.87 (0.78-1.01); Xs, 0.80 (0.66-0.93); filled squares, 0.57(0.31-0.72).

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to a method for preparing biologicalsamples, including collection and storage conditions, prior to testingfor ionized Mg²⁺ concentrations which allow accurate and reproduciblereadings. More specifically, the invention relates to a method ofcollecting and maintaining biological samples under conditions whichminimize or prevent exchange of gases between atmospheric air and thebiological sample, preferably in which O₂ is substantially excluded andpCO₂ levels are maintained in the biological sample prior to measuringionized Mg²⁺. The preferred embodiment is a method for preparing andstoring biological samples under anaerobic conditions.

The biological sample to be tested for ionized Mg²⁺ is preferably afluid or a sample that can be made fluid including but not limited towhole blood, plasma, serum, amniotic fluid, umbilical cord blood,cerebral spinal fluid, urine, gastric secretions, lacrimal secretions,peritoneal fluid, pleural fluid and the like obtained from animals,preferably mammals, most preferably humans. The fluid portion ofbiological tissue samples may be tested after homogenation with a tissuehomogenizer or the like if collected and maintained under conditionsdescribed herein. In the preferred embodiment, the biological sample iswhole blood, plasma, serum, cerebral spinal fluid, umbilical cord blood,and amniotic fluid.

Another aspect of the invention is a method for determining ionized Mg²⁺concentrations in a biological sample, collected and maintained underconditions which minimize or prevent exchange of gases between thebiological sample and atmospheric air, preferably in which O₂ issubstantially excluded and pCO₂ levels are substantially maintained inthe biological sample prior to measuring ionized Mg²⁺, most preferablyunder anaerobic conditions. In one embodiment, the ionized Mg²⁺concentration is measured using an ion selective electrode with aneutral carrier based membrane. In a preferred embodiment, the ionizedMg²⁺ concentration is obtained through the use of an ion selectiveelectrode manufactured by Nova Biomedical.

Another aspect of the invention is a method for determining ionized Ca²⁺:Mg²⁺ ratios in a biological sample, collected and maintained underconditions which minimize or prevent exchange of gases between thebiological sample and atmospheric air, preferably in which O₂ issubstantially excluded and pCO₂ levels are substantially maintained inthe biological sample prior to measuring ionized Ca²⁺ and Mg²⁺, mostpreferably under anaerobic conditions. In one embodiment, the ionizedCa²⁺ and Mg²⁺ concentrations are measured using ion selective electrodeswith a neutral carrier based membrane. In a preferred embodiment, theionized Ca²⁺ and Mg²⁺ concentrations are obtained through the use of anion selective electrode manufactured by Nova Biomedical.

Ion selective electrodes based on neutral carrier membranes and themethods of their use are known in the an and are widely used asintegrated devices in clinical chemistry analyzers. With theiravailability, a selective determination of different ions in dilutesamples as well as whole blood is possible (Oesch, U. et al. Clin. Chem32(8):1448, 1988). Neutral carrier based membranes selective forspecific ions are known in the an as described by Dinten, O. et al.(Anal. Chem. 63:596-603, 1991), which is incorporated by reference. Asexamples, membranes composed ofN,N'-diheptyl-N,N'-dimethyl-1,4-butanediamide (C₂₀ H₄₀ N₂ O₂) (ETH1117), N,N'-diheptyl-N,N'-dimethyl-aspandiamide (C₂₀ N₄₁ N₃ O₂) (ETH2220), N,N"-octamethylenebis(N'-heptyl-N'-methyl-2-methylmalondiamide)(C₃₂ H₆₂ N₄ O₄) (ETH 5214),N,N"-octamethylenebis(N',N'-dioctylmalondiamide) (ETH 5220),N',N",N"-iminodi-6,1-hexanediyldiiminotris(N-heptyl-N-methylmalonamide)(C₄₅ H₈₄ NO₆) (ETH 5282) and the like are known neutral carriersselective for Mg²⁺. Some examples of neutral carriers selective forCa.sup. 2+ are (-)-(R,R)-N,N'-[bis(11-ethoxycarbonyl)undecyl]-N,N',4,5-tetramethyl-3,6-dioxaoctanediamide (C₃₈ H₇₂ N₂ O₈) (ETH 1001),N,N,N',N'-tetracyclohexyl-3-oxapentanediamide (C₂₈ H₄₈ N₂ O₈) (ETH 129),N,N-dicyclohexyl-N',N'-dioctadecyl-3-oxapentanediamide (ETH 5234) andthe like. However, until the present invention, ion selective electrodesfor determining ionized magnesium concentrations were not known in theart.

The methods for determining total magnesium, ionized Ca²⁺ and Mg²⁺concentrations as measured using atomic absorption spectroscopy on anultrafiltrate are known in the art as described by Walser, M. (J. Clin.Invest. 40:723-730, 1961), D'Costa, M. (Clin. Chem. 29:5 19, 1983), andZaloga, G. P. et al. (Crit. Care Med. 15:813-816, 1987), which areincorporated by reference.

Another aspect of the invention is a method for diagnosing or prognosingdisease states or conditions associated with Mg imbalances, Mgdeficiencies, or Ca²⁺ :Mg²⁺ imbalances using a method of determiningionized Mg²⁺ or ionized Ca²⁺ :Mg²⁺ ratios in a biological sample,collected and maintained under conditions which minimize or preventexchange of gases between the biological sample and atmospheric air,preferably in which O₂ is substantially excluded and pCO₂ levels aresubstantially maintained in the biological sample prior to measuringionized Ca²⁺ and Mg²⁺, most preferably under anaerobic conditions. Suchdisease states include but are not limited to cardiac diseases,cardiovascular insufficiency, cardiac arrhythmias, coronary arteryspasm, those at risk for sudden death, renal disorders, lung diseases,respiratory muscle weakness, abnormal pregnancy, pre-eclampsia,eclampsia, fetal growth retardation, migraine, hypertension, idiopathicintracranial hypertension, diabetes, head trauma, premenstrual syndrome,tetany, seizures, tremor, apathy, depression, hypokalemia andhypocalcemia. The Mg²⁺ values of the patient are compared to normalionized Mg²⁺ values for biological samples. In one embodiment, thenormal ionized Mg²⁺ concentration in a normal adult is approximately0.53 to 0.67 mM, most preferably about 0.58-0.60 mM for the biologicalsample of whole blood, serum, and plasma. In another embodiment, thenormal ionized Mg²⁺ concentration is approximately 1.10-1.23 morepreferably 1.12-1.19 mM for the biological sample of cerebral spinalfluid.

A further aspect of the invention is a method of maintaining normalionized Mg²⁺ concentrations in a patient in need of such maintenance byadministering ionized Mg²⁺ in a concentration sufficient to maintainlevels of ionized Mg²⁺ in biological samples within a normal range ofionized Mg²⁺. In one embodiment, the normal ionized Mg²⁺ concentrationin a normal adult is approximately 0.53 to 0.67 mM, most preferablyabout 0.58-0.60 mM for the biological sample of whole blood, serum, andplasma. In another embodiment, the normal ionized Mg²⁺ concentration isapproximately 1.10-1.23 more preferably 1.12-1.19 mM for the biologicalsample of cerebral spinal fluid. Another aspect of the invention is amethod to attain or maintain normal ionized Mg²⁺ or Ca²⁺ and ionizedMg²⁺ concentrations in a patient in need of such maintenance byadministering Ca²⁺ and Mg²⁺ in a concentration sufficient to maintain anormal physiological molar ratio of ionized Ca²⁺ /ionized Mg²⁺ in theblood of about 1:1 to 2.5:1, more preferably about 1.5:1, and mostpreferably about 2:1, or to maintain a normal physiological molar ratioof ionized Ca²⁺ /ionized Mg²⁺ in the cerebral spinal fluid of about0.90:1 to about 1.15:1, more preferably 0.92:1 to about 1.1:1, mostpreferably about 1:1.

Another aspect of the invention is a method to attain or maintain normalionized Mg²⁺ or normal ionized Ca²⁺ and Mg²⁺ concentrations in aneonate, infant, and child in need of such maintenance by administeringMg²⁺ alone or in combination with Ca²⁺ in a concentration sufficient tomaintain a normal physiological molar ratio ionized Ca²⁺ /ionized Mg²⁺in the blood of about 1.9:1 to about 2.6:1, more preferably about 2.3:1to about 2.5:1.

An additional aspect of the invention is a composition for use inpreventing or treating magnesium imbalances, magnesium deficiencies orCa²⁺ :Mg²⁺ imbalances, the composition is composed of bioavailablemagnesium alone or in combination with bioavailable calcium in aneffective concentration. The concentration of calcium and magnesium is aconcentration that provides to an individual a normal physiologicalmolar ratio of ionized calcium to ionized magnesium in the blood ofabout 1:1 to 2.5:1, more preferably about 1.5:1, and most preferablyabout 2:1, or to maintain a normal physiological molar ratio of ionizedCa²⁺ /ionized Mg²⁺ in the cerebral spinal fluid of about 0.90:1 to about1.15:1, more preferably 0.92:1 to about 1.1:1, most preferably about1:1. In another embodiment the concentration of calcium and magnesium isa concentration that provides to a neonate, infant and child a normalphysiological molar ratio of ionized calcium to ionized magnesium in theblood of about 1.9:1 to about 2.6:1, more preferably about 2.3:1 toabout 2.5:1. The composition is useful in treating individuals with thefollowing disease states such as cardiac diseases, hypertension,idiopathic intracranial hypertension, diabetes, lung diseases, abnormalpregnancy, preeclampsia, eclampsia, head trauma, fetal growthretardation or other diseases associated with magnesium deficiencies oran imbalance of ionized Ca²⁺ /ionized Mg²⁺ ratios in the whole blood,plasma, serum, cerebral spinal fluid or the like.

The use of the composition is not limited to individuals with theaforementioned diseases but may also be used in healthy individual formaintaining proper ionized magnesium or ionized calcium/ionizedmagnesium concentrations. Such maintenance is useful in preventingmagnesium imbalances, Ca²⁺ /Mg²⁺ imbalances, or magnesium deficienciesand in turn is useful in preventing magnesium-associated disease states.

The composition may be taken alone as a therapeutic agent in apharmaceutically acceptable carrier or a mineral supplement, or thecomposition may be added to supplement other ingredients such as, butnot limited to, vitamin formulations, vitamin and mineral formulations,and foodstuffs. Such food stuffs include solids and liquids. In oneembodiment the composition is added to infant formulas.

EXAMPLE I SAMPLE COLLECTION

In order to obtain precise and reproducible determinations of ionizedMg²⁺ with the ISE, blood samples were collected under conditions thatminimize or prevent exchange of atmospheric gases with those of thesample, most preferably under anaerobic conditions into a tube with theair evacuated, such as a Vacutainer™ tube, or other tube or syringesubstantially free of atmospheric gases, especially O₂. The tube orsyringe may contain heparin (<75u/ml, more preferably <50/ml, mostpreferably <20u/ml). After collection of the biological sample, thesamples are placed in and kept under conditions that minimize or preventexchange of atmospheric gases with those of the sample, most preferablyunder anaerobic conditions (FIG. 1).

EXAMPLE II SAMPLE PROCESSING

To process clotted blood or plasma (heparinized blood), the samples weremaintained under conditions that minimize or prevent exchange of gasesbetween atmospheric air and the biological sample, preferably anaerobicconditions in tubes sealed with rubber stoppers. Parafilm or plastic andglass tops cannot be utilized as this allows for air to enter thesample. If samples (i.e., whole blood, serum or plasma) were analyzed(or frozen) more than 30 min after blood draw for processing of serum orplasma, the sealed tubes were placed in a standard clinical orlaboratory centrifuge and centrifuged at 3,000-4,000 rpm for 15-20minutes. After this time, the sera or plasma was carefully removed fromthe packed cells by inserting a sterilized needle attached to either aplastic syringe (for sera) or a lightly heparinized (<75u/ml, morepreferably <50u/ml, most preferably <20u/ml) glass syringe (for plasma)or a similar device.

EXAMPLE III SAMPLE STORAGE

The anaerobically maintained samples were either processed with the ISEwithin six hours or the plasma or serum carefully expelled into a tubewith the air evacuated or other tube or syringe substantially free ofatmospheric air, especially O₂, most preferably anaerobic, with orwithout heparin, and frozen at -10° C. Unlike ionized Ca²⁺, ionized Mg²⁺levels were stable during numerous subsequent freeze-thaw procedures,provided the bloods were drawn and processed under anaerobic conditionsas described above (see FIG. 2, Table 1). Under conditions whereparafilm coverings were used, pH and pCO₂ changes occurred causingerratic and erroneous ionized Mg²⁺ values.

                  TABLE 1                                                         ______________________________________                                        Influence of Freeze-Thawing with Parafilm Covering on Plasma                  pH and Ionized Free Mg.sup.2+  Obtained with an Ion Selective                 Electrode                                                                             Day of Examination                                                    Parameter 1         2         3      12                                       ______________________________________                                        pH        7.435 ±                                                                              7.685 ±                                                                              7.897 ±                                                                           8.26 ±                                          0.010     0.044     0.056  0.070                                    Mg.sup.2+ 0.64 ± 0.65 ± 0.60 ±                                                                            0.53 ±                                          0.035     0.04      0.029  0.036                                    ______________________________________                                    

Using the method of the present invention, plasma and serum samples werefrozen for up to two weeks at -10° C. without affecting the ionized Mg²⁺values. Whole blood samples were maintained under anaerobic conditionsat room temperature for up to six hours after blood draws prior toionized Mg²⁺ determinations. These and other experiments clearlyindicated that as the biological samples such as blood, sera or plasmabecame exposed to air, they loose CO₂, and as a consequence the pHbecame alkaline. This adversely affected the ionized free Mg²⁺ valuesresulting in erroneous readings. This is completely obviated with properhandling as describe for this invention.

EXAMPLE IV SAMPLE PREPARATION FOR PRECISION, SPECIFICITY AND INTERFERANTSTUDIES

Male and female subjects that had no electrolyte abnormalities, agesranging from 19-83 years, were used for the reference range study.

Whole blood and plasma samples were collected using a needle attached toheparinized Vacutainer tubes; serum from-red top Vacutainer tubes. Allblood samples were collected and maintained under anaerobic conditionsand processed within 1-2 hours of collection. Ultrafilterable Mg wasobtained using an Amicon micropartition system (3,000 MW cutoff) aftercentrifugation of the plasma or serum at 1,500 to 2,000 RCF (g) for 20minutes. A 3,000 MW cutoff was utilized in order to retain smallmolecular wt peptides. However, when normal sera from six volunteerswere processed using a 30,000 MW cutoff virtually identical results wereobtained.

Precision (within run, day-to-day) was determined on three levels ofaqueous control solutions obtained from NOVA Biomedical containing115,135 and 155 mM Na⁺ ; 2.0, 3.75, and 5.75 mM K⁺ ; 0.50, 1.00 and 1.50mM Ca²⁺ ; and 0.30, 0.50, and 1.00 mM Mg²⁺ at pH values of 7.15, 7.35,and 7.58, respectively.

Aqueous solutions of MgCl₂ were examined for the linearity study andaqueous solutions of various cations (Ca²⁺, K⁺, Na⁺, H⁺, NH₄ ⁺, Fe³⁺,Cu²⁺, Zn²⁺, Cd²⁺, Hg²⁺, and Pb²⁺) were examined over pathophysiologicconcentration ranges for potential interference to the new Mg²⁺electrode. The concentrations of potentially interfering cations werechosen based on the following observations: the upper limit of thereference range (ULRR) for NH₄ ⁺ is <100 μM; the toxic range for Cd²⁺has been listed up to 27 μM; concentrations for Ca²⁺ rarely exceed 2.0mM; the ULRR for Cu²⁺ is <30 μM; for Fe²⁺ <50 mM; for Pb²⁺ <2 μM; forHg²⁺ <0.25 μM; and for Zn²⁺ 23 μM. K⁺ above 10 mM and Na⁺ above 175 mMare rarely seen in plasma. Ligand binding studies were also carried outin aqueous solutions containing pathophysiological concentrations ofheparin, lactate, acetate, phosphate, bicarbonate and sulfate.

Albumin/pH studies employed bovine serum albumin (Sigma Chem. Co., St.Louis, Mo.) that was treated with an union-exchange resin (ExchangeResin AG50W-48, Bio-Rad Richmond, Calif.) resulting in a solution thathad 6 g albumin/dl and unmeasurable TMg (<0.04 mM, as assessed by atomicabsorption spectrophotometry). This solution was lyophilized followed bygravimetric addition of lyophylate to aliquots of an aqueous solutioncontaining a fixed amount of Mg.

Spiking experiments with plasma (pH 7.6-7.7) and whole blood (pH7.40-7.44) were also performed with addition of various concentrationsof mostly MgCl₂ and some with MgSO₄ • 7H₂ O (Biological Grades, ACScertified, Fisher Scientific, New Jersey) at room temperature. Electrodeanalyses for IMg²⁺ results were performed immediately after beingwell-mixed, and after 5 and 10 min; results were identical at all threetime intervals.

All chemicals used to make up the aqueous solutions were of high purity(biological, ACS certified grades) and obtained form Fisher ScientificCo., and Sigma Chemical Co.

For most aqueous solutions, a 5 mM HEPES buffered-physiologic saltsolution (in Mm/L) (120-140 NaCl; 4-5 KCl; 1 CaCl₂) was used. In somecases, due to increased acidity, caused by certain ligands, 10 mM HEPESwas added to the latter instead of 5 mM HEPES. In the case of the pHstudies, modified KREBS-Ringer bicarbonate buffered physiological saltsolutions were used (in mM/L) (118 mM NaCl; 4.7 KCl; 1.2 KH₂ PO₄ ; 1.0CaCl₂ ; 25 NaHCO₃) gassed with a 5% CO₂ -95% O₂ mixture.

A NOVA Stat Profile 8 (SPS) Analyzer (NOVA Biomedical, Waltham, Mass.)containing the specially-designed Mg²⁺ electrode along with electrodesfor Ca²⁺, Na⁺, K⁺, and pH, and, where appropriate, hematocrit, was usedfor these studies.

The electrode is calibrated by using two aqueous solutions containingdifferent concentrations of MgCl₂ in the presence of known pH andconcentrations of Na⁺, K⁺, and Ca²⁺. The values assigned to thesesolutions are determined gravimetrically. The electrical signal from theMg²⁺ electrode is mathematically adjusted by the signal from the Ca²⁺electrode to provide the resulting Mg²⁺ concentration. All electrodemeasurements can be made on a 250 μl sample (whole blood, plasma orserum) within 90-120 seconds. The SP8 is equipped with its own on-boardcalibrators.

Total magnesium values are obtained on a Perkin-Elmer Model Zeeman 5000atomic absorption spectrophotometer (AAS), utilizing 1% LaCl₃ (FisherScientific) to prevent any interferences.

Data were evaluated for statistical significance using means ±S.E.M.,unpaired t-tests, ANOVA, method of least squares for regression analysesand correlation coefficients, where appropriate. A p value less than0.05 was considered significant.

EXAMPLE V DETERMINATION OF MAGNESIUM IN AQUEOUS SOLUTIONS: ISECOMPARISON TO ATOMIC ABSORPTION

FIG. 3 illustrates that the Mg²⁺ electrode quantifies Mg comparably toatomic absorption spectroscopy in aqueous solution. In the absence ofbinding ligands, it would be predicted that the values from the twotechnologies would be identical. Results from these measurements werelinear over the entire 0.1 to 3.0 mM Mg²⁺ range studied.

EXAMPLE VI PRECISION AND REPRODUCIBILITY

Using the ISE on three levels of aqueous Mg²⁺ controls, mean values overa range of 0.3 to 1.0 mM are within 94.6 and 99.2% of their targets. Thelinearity of the ISE (0.1-3.0 mM) in aqueous solution and human plasmaand serum ranges between 92.0 and 99.3%.

EXAMPLE VII EFFECTS OF ALBUMIN AND pH ON MEASURED IMg²⁺ LEVELS

The effect of protein on measured IMg²⁺ levels were strongly affected bythe pH of the medium. Results in Table 2 show that, in unbufferedsolutions, addition of albumin up to a final concentration of 150 g/lproduces a moderate decline in measured IMg²⁺ values. The observeddecline in pH of the medium upon addition of albumin is expected sincethe protein has a acidic isoelectric point. If, however, the pH of thesolution is carefully alkalinized by addition of NaOH, measured IMg²⁺values decline by up to 75%, indicating that the exposure of anionicgroups on albumin promotes the binding of Mg²⁺.

                  TABLE 2                                                         ______________________________________                                        Adding Albumin to an Aqueous Mg Solution Followed by                          Alkalinization                                                                Albumin added      Mg.sup.2+                                                                             Mg.sup.2+  (mM After adjusting                     (g/L)      pH      (mM)    pH to 8.24 ± 0.03)                              ______________________________________                                        0          7.45    0.49                                                       30         7.02    0.44    0.39                                               60         6.67    0.41    --                                                 90         6.37    0.40    0.20                                               120        6.15    0.40    0.17                                               150        5.98    0.43    0.13                                               ______________________________________                                    

To differentiate a possible direct effect on pH on the performance ofthe electrode, measurements over a similar range of pH values (bufferedby phosphate and/or bicarbonate) were performed in the absence ofalbumin. Results in FIG. 4 show that IMg²⁺ measurements are minimallyaffected over the pH range examined (6.2 to 8.5). Only at the lower pHvalues were measurements of IMg²⁺ levels affected (10-20%). Thesestudies, thus, show that over the pH ranges examined, pH per se does notsignificantly influence the measured IMg²⁺ values. H⁺ clearly affectsICa²⁺ much more than IMg²⁺, causing an apparent 140% decrease in ICa²⁺over the pH range of 6.3 to 8.3. This is likely due to the precipitationof calcium compounds.

EXAMPLE VIII EFFECTS OF COMMON CATIONS AND HEAVY METAL IONS ON IMg²⁺VALUES

Virtually no interference was noted from pathophysiologic concentrationsof any of the cations added to an aqueous solution containing Mg²⁺ withthe possible exception of Zn²⁺ (Table 3). Adding calcium salts to serumcaused an increased in the IMg²⁺ (˜0.1 mM Mg²⁺ for a 1.0 mM increase inCa²⁺), but a much smaller increase was seen when calcium was added to anaqueous solution containing Mg²⁺ (˜0.01-0.04 mM Mg²⁺ for a 1.77 mMincrease of Ca²⁺). The change in plasma IMg²⁺ is likely the result ofadded Ca²⁺ competing for binding sites held by Mg²⁺ in the plasma. Theconcentration of NH₄ ⁺ and heavy metal ions represent the upper limit oftheir reference range, except for Cd²⁺, whose concentration was toxic.

                                      TABLE 3                                     __________________________________________________________________________    Influence of Common Cations and Heavy Metal Ions on the Mg.sup.2+             Determined                                                                    by ISE                                                                        Final                      Mg.sup.2+  (mM) in the                             Cation conc.                                                                             Mg.sup.2+  (mM)                                                                       Final Cation                                                                          aqueous solution                                   Cation                                                                            in plasma                                                                            in plasma                                                                             conc. in water                                                                        Soln. 1                                                                            Soln. 2                                       __________________________________________________________________________    NH.sub.4.sup.+     0    mM 0                                                                     1       1                                                  Cd.sup.2+                                                                         0  μM                                                                             1.25 ± 0.050*                                                       57     1.24 ± 0.051                                                    Ca.sup.2+                                                                         0.75                                                                             mM  1.02 ± 0.003                                                                       1.02-1.32                                                                          mM 0.50 0.56                                              1.50   1.01 ± 0.010                                                                       1.46-1.48                                                                             0.50 0.57                                              1.90   1.07 ± 0.011                                                                       1.87-2.79                                                                             0.51 0.58-0.60                                     Cu.sup.2+                                                                         0  μM                                                                             0.94 ± 0.025                                                        24     0.94 ± 0.010                                                    Fe.sup.3+                                                                         0  μM                                                                             1.25 ± 0.050                                                        28.6   1.23 ± 0.020                                                    Pb.sup.2+          0    μM   0.50                                                             1.93         0.51                                          Hg.sup.2+                                                                         0  μM                                                                             0.88 ± 0.015                                                        0.10   0.88 ± 0.015                                                    K.sup.+                                                                           4.18                                                                             mM  1.02 ± 0.005                                                                       0    mM      0                                                 10.00  0.97 ± 0.005                                                                       5            0                                             Na.sup.+                                                                          145                                                                              mM  1.01 ± 0.00                                                                        0    mM      0                                                 175    1.02 ± 0.003                                                                       75           0.01                                          Zn.sup.2+                                                                         0  μM                                                                             0.91 ± 0.020                                                        10     0.91 ± 0.019                                                        18     0.99 ± 0.050                                                    in serum (μM)                                                              0          0.57                                                               25         0.61                                                               __________________________________________________________________________     *Values are means ± S.E.M.                                            

EXAMPLE IX SMALL LIGAND BINDING TO Mg²⁺

It appears that plasma samples having a heparin concentration of 20units/ml or less will produce less than a 4% error for the IMg²⁺determination (Table 4). Several small ligands (e.g., acetate,bicarbonate, citrate, lactate, phosphate, sulfate), on the other hand,may bind significant amounts of Mg²⁺, suggesting that the IMg²⁺ /TMgratio could vary within a patient over time, in an acute-care settingdepending on the solutions the patient is receiving.

                  TABLE 4                                                         ______________________________________                                        Performance of Mg.sup.2+  ISE in Presence of Various Ligands                  in Aqueous Solution                                                                         Average [Mg.sup.2+ ]                                                                       Average                                                                          Location                                                                             % change                                 Ligand  Concentrations                                                                            Location 1                                                                              2      in [Mg.sup.2+ ]                          ______________________________________                                        acetate 0      mM       1.04 2.22 1.03                                                0.5             0.99 2.20 1.03   -2                                           20.0            0.92 1.86 0.95   -12                                  bicarbonate                                                                           0      mM       1.09 2.09 1.03                                                10              1.01 1.96 1.07   -3                                           40              0.94 1.85 0.99   -10                                  citrate 0      mM       1.18 2.19                                                     0.5             0.88 1.80        -22                                          20.0            0.08 0.17        -94                                  heparin 0.0    U/ml     1.05 1.96 1.03                                                10                        1.07   +4                                           20              0.97 1.94 1.01   -4                                           50              0.90 1.89        -9                                           100             0.83 1.71 0.95   -14                                          500             0.50 1.07        -49                                  lactate 0      mM       0.97 2.19                                                     0.5             0.92 1.98        -8                                           20.0            0.76 1.64        -24                                  phosphate                                                                             0      mM       1.06 2.02 1.03                                                1               1.06 1.90 1.03   -2                                           2               0.95 1.69        -13                                          5                         0.81   -21                                  sulfate 0      mM       1.05 2.02 1.03                                                0.1             1.04 1.98 1.03   -1                                           1.0             0.99 2.00 1.01   -3                                           10.0            0.93 1.80        -11                                  ______________________________________                                    

EXAMPLE X REFERENCE RANGES

Assessment of ionized Mg²⁺ in whole blood, plasma and serum with the ISEindicate that ionized Mg²⁺ is held within an extremely narrow range(0.53-0.67 mM, mean=0.58±0.0065, n=60) when compared to total Mg(0.70-0.96 mM, mean=0.81 mM±0.0084) or ionized Ca²⁺ (1.09-1.30 mM). Thisnarrow range for ionized Mg²⁺ has not previously been reported. Thesedata were derived from approximately 60 normal healthy human subjects.The IMg²⁺ /TMg ratio in this group ranged from 61-85% with a mean of71.6±0.58%. The mean value for ionized Mg²⁺ is approximately one-half ofwhat it is for ionized Ca²⁺ and thus, represents a ICa²⁺ /IMg²⁺ ratio inhuman blood (plasma or serum) of about 2.0. Such a narrow range forionized Mg²⁺ obtained with the ISE suggested that slight changes in thenormal ionized Mg²⁺ range could be diagnostic and prognostic fornumerous pathophysiologic states and disease conditions in animals andhuman subjects (Altura et al. Clinical Res., in press; Handwerker, S. etal. Magnesium and Trace Elements, in press; Altura et al. Magnesium andTrace Elements, in press).

EXAMPLE XI COMPARISON OF Mg LEVELS IN WHOLE BLOOD, PLASMA, SERUM ANDTHEIR ULTRAFILTRATES

A comparison of the measured levels of TMg and IMg²⁺ in whole blood,plasma and serum are shown in Table 5. All values shown are based onanalysis of samples collected from healthy volunteers, with theexception of serum samples having an n value of 237, which includedsamples collected from patients undergoing cardiac surgery.

                                      TABLE 5                                     __________________________________________________________________________    Regression Analysis of Serum, Plasma and Whole Blood Ionized Mg.sup.2+        and Total Mg                                                                                            y      x                                            Regression of y on x                                                                     n  r  Slope                                                                             Intercept                                                                          Mean                                                                              SD Mean                                                                              SD                                       __________________________________________________________________________    STMg.sup.a on PTMg.sup.a                                                                 21 0.81                                                                             0.63                                                                              0.30 0.83                                                                              0.06                                                                             0.84                                                                              0.08                                     SMg.sup.2+b on PTMg.sup.2+b                                                              21 0.93                                                                             0.89                                                                              0.08 0.59                                                                              0.05                                                                             0.58                                                                              0.06                                     PMg.sup.2+b on PTMg.sup.a                                                                74 0.80                                                                             0.55                                                                              0.13 0.56                                                                              0.06                                                                             0.78                                                                              0.08                                     SMg.sup.2+b on STMg.sup.a                                                                237                                                                              0.88                                                                             0.71                                                                              0.01 0.74                                                                              0.20                                                                             1.039                                                                             0.25                                     WBMg.sup.2+b on PMg.sup.2+                                                               18 0.79                                                                             1.02                                                                              -0.02                                                                              0.57                                                                              0.05                                                                             0.57                                                                              0.06                                     __________________________________________________________________________     STMg = serum total Mg                                                         PTMg = plasma total Mg                                                        SMg.sup.2+  = serum ionized Mg                                                PMg.sup.2+  = plasma ionized Mg                                               WBMg.sup.2+  = whole blood ionized Mg                                         .sup.a = Assessed by atomic absorption spectroscopy                           .sup.b = by ISE for Mg.sup.2+                                            

The highest correlation obtained (0.93) was for comparisons of IMg²⁺ inplasma and serum samples. Interestingly, a significantly (p=0.02) lowercorrelation (r=0.79) was observed when a similar comparison was madebetween IMg²⁺ levels in plasma vs. whole blood. The greater varianceobserved may suggest that a slight redistribution of bound Mg²⁺ occursupon removal of formed blood elements. The lower values of r, seen forcomparisons of TMg to IMg²⁺ in serum and plasma samples indicates that arange of IMG²⁺ values exist for a given level of TMg. This is clearlyseen in FIG. 5 which is a plot of serum TMg vs. IMg²⁺ values shown inTable 5. A greater range of IMg²⁺ values are seen in this plot, as manyof the samples were from patients in whom cardioplegia was induced usingMg²⁺ -supplemented solutions.

Results of measurements of IMg²⁺ and TMg performed on ultrafiltrates ofserum and plasma and neat-samples are shown in Table 6. Plasma samplesare collected from healthy volunteers, whereas serum samples alsoincluded ones from the cardiac patients. A comparison of mean values forTMg demonstrates that plasma and serum protein-free filtrates have TMglevels of 65% (0.55/0.84×100) and 71% (0.66/0.93×100), respectively, ofthese measured in the neat-samples. These values are similar to IMg²⁺levels measured in the neat-samples. Subsequent measurements onultrafiltrates with the ISE, however, yielded values that were only 79%(0.45/0.57×100) and 85% (0.64/0.75×100) respectively, of the filteredTMg levels in these samples. These lower levels indicate that lowmolecular weight compounds are present which can chelate Mg²⁺, renderingthis fraction insensitive to the ISE. The size of this fraction,however, is likely somewhat overestimated by these measurements as thepH of the ultrafiltrates was always greater than the starting pH valueof the unfiltered samples by 0.6-1.1 units, thereby reducing competitionbetween H⁺ and Mg²⁺ for binding to these agents.

                                      TABLE 6                                     __________________________________________________________________________    Regression Analysis of Total Mg and Ionized Mg.sup.2+  in Plasma, Serum       and Their                                                                     Ultrafiltrates                                                                                          y      x                                            Regression of y on x                                                                     n  r  Slope                                                                             Intercept                                                                          Mean                                                                              SD Mean                                                                              SD                                       __________________________________________________________________________    uPTMg on PTMg                                                                            30 0.77                                                                             0.51                                                                              0.13 0.55                                                                              0.05                                                                             0.84                                                                              0.07                                     uSTMg on STMG                                                                            48 0.93                                                                             0.66                                                                              0.03 0.66                                                                              0.20                                                                             0.93                                                                              0.28                                     PMg.sup.2+ 30n uPTMg                                                                        0.69                                                                             0.86                                                                              0.11 0.58                                                                              0.05                                                                             0.55                                                                              0.05                                     SMg.sup.2+ 48n uSTMg                                                                        0.97                                                                             0.99                                                                              0.02 0.67                                                                              0.20                                                                             0.66                                                                              0.20                                     uPMg.sup.2+  on uPTMg                                                                    10 0.64                                                                             0.37                                                                              0.24 0.45                                                                              0.03                                                                             0.57                                                                              0.05                                     uSMg.sup.2+  on uSTMg                                                                    25 0.94                                                                             0.90                                                                              -0.04                                                                              0.64                                                                              0.21                                                                             0.75                                                                              0.21                                     __________________________________________________________________________     uPTMg = ultrafilterable plasma total Mg                                       uSTMg = ultrafilterable serum total Mg                                        uPMg.sup.2+  = ultrafilterable plasma Mg.sup.2+  assessed with ISE            uSMg.sup.2+  = ultrafilterable serum Mg.sup.2+  with ISE                 

The increase in scatter between TMg²⁺ and IMg²⁺ going from FIG. 3(aqueous solutions) to FIG. 5 (patients samples) results fromdifferences in Mg binding to ligands and proteins from sample to sample.However, the ratio of IMg²⁺ /TMg was remarkably similar, averaged across74 plasma samples and across 237 serum samples; 71% in both cases (Table5) even though the IMg²⁺ /TMg ratio for individual patients differentsignificantly from the average. This suggests that 29% of the TMg wastypically bound to small anions and/or proteins in the "normal" and theCPB patient populations included in this study. These results suggestless binding of Mg to protein then has been described in the literature;33-34% protein-bound by Speich et al. (Clin. Chem. 1981.27:246-248) and32% protein-bound by Kroll et al. (Clin. Chem. 1985.31:244-246).

The present ultrafiltration studies raise questions in terms of what Mgfraction(s), or portions thereof, the ultrafiltrate actually represents.It has long been thought that the ultrafiltrate is really the plasma (orserum) minus only large molecular weight proteins with their bound Mg(Aikawa, J. K., Magnesium, Its Biological Significance Boca Raton: CRCPress, 1981; Waser, M. 1967. Rev. Physiol. Biochem. Exp. Pharmacol59:185-341; Elin, R. J. Clin. Chem. 1987.33:1965-1970). However, it islikely that the pH increase (from 0.6-1.1 pH units) over the course ofthe ultrafiltration process, itself, caused additional protein bindingof Mg²⁺. Such increased binding is supported by the fact that IMg²⁺measured values were reduced by alkalinizing protein-containingsolutions of Mg²⁺ to higher pH values. Thus, at the lower H⁺concentrations, the total concentration of Mg in the ultrafiltrate isless than it would have been at pH 7.4. In addition, it is clear thatnot all the Mg in the ultrafiltrates is ionized. Twenty-one percent ofthe plasma ultrafiltrate Mg is bound [ (0.57-0.45)/0.57×100)].

The percentage of Mg²⁺ bound to ligands and protein may remainremarkably constant for a given patient, albeit a from the typical ratioseen across all patients, even when TMg is changing markedly such as incoronary bypass patients; or the percentage bound may change appreciablywithin hours. The total Mg concentration or the ultrafilterable Mgconcentration in a given sample thus cannot be used to predict the levelof ionized Mg²⁺ in the plasma or serum. This clearly indicates thatmonitoring the ionized Mg²⁺ level rather than total magnesium or theultrafilterable Mg concentration is a valuable diagnostic and prognosticparameter in critical care and acute media care settings.

EXAMPLE XII SPIKING Mg INTO PLASMA AND WHOLE BLOOD SAMPLES

Results in Table 7 demonstrate the effect of adding Mg²⁺ (Cl⁻ or SO₄ ²-salt) to samples of plasma and whole blood. As expected, for plasmasamples, the calculated recovery values are less than 100% indicatingthat added Mg²⁺ is partially bound to plasma proteins. The valuesreported have not been corrected for displacement of plasma water byprotein and lipids. The observation that the fraction of recovered Mg²⁺increases slightly with additional amounts of added Mg²⁺ indicates thatpartial saturation of anion binding sites is likely occurring. Forexperiments with whole blood, a fixed amount of Mg²⁺ was added (1.0 mM).The recovery levels observed with whole blood were considerably higherthan would be predicted by calculating molarity based on whole bloodvalue. This is attributed to the fact that the added magnesium saltinitially dissolves in the plasma water volume of the sample rather thanbeing evenly distributed throughout the entire sample. The largedifferences seen in the spiked samples may reflect differences inhematocrit or possibly the time between Mg²⁺ addition and when thesample was analyzed. In addition, differences may also exist beensamples in the binding capacity by plasma proteins and solutes.

                                      TABLE 7                                     __________________________________________________________________________    Spiking of MgCl.sub.2 or MgSO.sub.4 into Pooled Plasma and Fresh Whole        Blood                                                                         Pooled Plasma (0.56 mM IMg.sup.2+)                                                                    Fresh Whole Blood                                     Mg.sup.2+                                                                         Ideal               (0.51-0.63 mM IMg.sup.2+)                             Added                                                                             Expected                                                                             Measured                                                                             %     Ideal Expected                                                                        Measured                                                                             %                                      (mM)                                                                              Value (mM)                                                                           Value (mM)                                                                           Recovery                                                                            Value (mM)                                                                            Value (mM)                                                                           Recovery                               __________________________________________________________________________    0.5 1.06   0.97 ± 0.048*                                                                     82.0  --      --     --                                     1.0 1.56   1.35 ± 0.027                                                                      79.0  1.57 ± 0.017                                                                       2.20 ± 0.154                                                                      163.0                                  1.5 2.06   1.78 ± 0.052                                                                      81.3  --      --     --                                     2.5 3.06   2.68 ± 0.103                                                                      84.8  --      --     --                                     __________________________________________________________________________     N = 3-6 experiments each                                                      *+Results are means ± S.E.M.                                          

EXAMPLE XIII COMPARING IMg²⁺ TO TMg IN CPB PATIENTS

Results shown in FIG. 6 are subset of data from FIG. 5 which showed thatthe level of TMg in a given sample cannot be used to predict IMg²⁺.Results plotted in FIG. 6 are TMg and IMg²⁺ values for several samplesfrom each of three patients over the course of their cardiac surgeries.The first two samples from each patient, taken before cardioplegiacontaining Mg²⁺ was administered, had values near the regression linetaken from the data in FIG. 6. Following induction of cardioplegia,levels of TMg and IMg²⁺ increase significantly (arrows left to right)while subsequent samples demonstrated a gradual decrease in bothparameters (arrows to the left). These results show that not only didthe ratio of IMg²⁺ /TMg differ between three patients, but it remainedrelatively constant within a patient during the course of theperioperative period. In other patients, the IMg²⁺ /TMg ratio changedsignificantly during this period.

EXAMPLE XIV CELLULAR AND EXTRACELLULAR DISTRIBUTION OF MG

Having the information on the precise concentrations of extracellularionized Mg in human blood allows one to determine the cellular andextracellular distribution of Mg. Using ³¹ P-nuclear magnetic resonancespectroscopy and digital image analysis on cardiac myocytes, vascularsmooth muscle cells and intact brain, the intracellular free ionizedMg²⁺ was determined to be approximately 600-700 micromolar (Altura, B.M. et al. Influence of Mg²⁺ on Distribution of Ionized Ca²⁺ in VascularSmooth Muscle and on Cellular Bioenergetics and Intracellular Free Mg²⁺and pH in Perfused Hearts Probed by Digital Imaging Microscopy. In:Imaging Technique in Alcohol Research, S. Zhakari, H. Witt (Eds.),NIAAA; Wash., D.C. Gov't Printing Office, 1992; Altura, B. M. et al. J.Magn. Reson. Imaging, 1992, in press; Barbour, et al. FASEB J. 1989, 3:A250; Barbour, et al. Magnesium and Trace Elem., 1992, in press; Zhang,A. et al, Biochem, Biophys. Acta Mol. Cell Res. 1992, in press).Concentrations of ionized Mg²⁺ across mammalian cell membranes werequite similar, that is about 500-600 micromolar. Although the relativeconcentration of IMg²⁺ outside cells is about 71% of the totalextracellular Mg, the relative amount of intracellular free ionized Mgis much less, only about 3%-6%.

EXAMPLE XV DIAGNOSIS & PROGNOSIS IN CARDIOVASCULAR DISEASE

It has been suggested that abnormalities in Mg metabolism may play animportant role in the etiology of cardiac diseases (Altura, B. M. Drugs28 (Suppl. I): 120-142, 1984; Altura, B. M. et al. In: Metal Ions inBiological Systems Vol 26, 1990; Iseri, C. T. West. J. Med. 138:823-828,1983; Ebel, H. et al. J. Clin. Chem. Clin. Biochem. 21:249-265, 1983;Altura, B. M. et al. Magnesium 4:226-244, 1985; Sjogren, A. et al. J.Intern Med. 226:213-222, 1989; Zaloga, G. P. et al. In: Problems inCritical Care Vol 4, 1990; Rudnick, M. et al. APMIS 98:1123-1127, 1990).Although elevated extracellular Mg²⁺ is widely used in connection withcardiopulmonary bypass (CPB) procedures, it is not known whether suchprocedures result in rapid and sequential alterations in blood ionizedMg²⁺ levels. By using the methods of the present invention, ionizedMg²⁺, along with ionized Ca²⁺ levels in plasma were monitored inpatients prior to, during, and after CPB. The patients studies ranged inage from 10-80 yrs. and were scheduled for coronary bypass, valvereplacement or other elective open-heart procedures (OHP). On the basisof studies with 30 human subjects prior to and during cardiopulmonarybypass (CPB), subjects had lower than normal ionized Mg²⁺ prior tosurgery (Altura, B. T. et al. Clin. Chem. (Jul-Aug), 1991; Altura, B. T.et al. Clin. Res., in press; Altura, B. T. et al. Magnesium and TraceElements, in press). Assessment of ionized Mg²⁺ in plasma revealed thefollowing [means±S.E.M. in millimolar conc. (mM)]: prior toOHP=0.56±0.03 vs. 0.60±0.005 (control); within 10-15 minutes ofCPB=0.89±0.08; post perfusion=0.75±0.03. In addition, on the basis offrequent determinations during CPB, using an ion selective electrode thedegree of spontaneous hypotension, arrhythmias, and coronary vasospasmduring and post-surgery were correlated to the pronounced alterations inionized Ca²⁺ :ionized Mg²⁺ ratios. (Altura, B. T. et al. Clin. Res., inpress; Altura, B. T. et al. Magnesium and Trace Elements, in press).With respect to ionized Ca²⁺, the respective values were 0.96±0.016 vs.1.21±0.01; 0.79±0.02; and 1.23± 0.10. Although the normal ionized Ca²⁺:ionized Mg²⁺ ratio is 1.95-2.18, all patients studied prior to CPBexhibited lower values (mean=1.72±0.09). Within 10-15 minutes of initialCPB, the ionized Ca²⁺ :ionized Mg²⁺ ratio fell almost 50%(mean=0.91±0.10); post-perfusion, the ratio rose to 1.62±0.18. Overall,these data indicate that ionized Mg²⁺ concentrations can be monitored inplasma during CPB. Predictable patterns arose out of these studies,showing that cardiac disease patients tend to exhibit lower than normalionized Mg²⁺, ionized Ca²⁺ and ionized Ca²⁺ :ionized Mg²⁺ ratios.Additionally, the hypotension observed upon initiation of CPB may inpart be a reflection of elevated ionized Mg²⁺ and a pronounced drop inthe ionized Ca²⁺ :ionized Mg²⁺ ratio. Such patterns are thereforediagnostic and predictive, thus allowing the physician and surgeon tocarefully monitor and treat such cardiac patients.

EXAMPLE XVI DIAGNOSIS & PROGNOSIS IN HEAD TRAUMA

The present studies were undertaken to determine if head trauma wasassociated with deficits in serum, plasma and whole blood ionized Mg²⁺and to determine if the degree of head injury would correlate with thedegree of the plasma ionized Mg2+ deficiency. Head trauma patientsclearly demonstrated that head trauma and the degree of head trauma wasassociated with deficiencies in ionized Mg²⁺. The range of ionized Mg²⁺in plasma of these head trauma cases was significantly below normal; thegreater the degree of head trauma (as assessed by clinical signs andGlasgow scores), the greater the deficit in ionized Mg²⁺.

Sixty-six patients (male=44; females=22), presented in the emergencyroom of a large community hospital; ranging in age from 12-83 yrs., werestudied. Patients with blunt head trauma were studied within 1-8 hrs ofthe event and compared with 60 age-matched controls and 14 patientcontrols with minor peripheral trauma such as cuts and sprains. A groupof normal, healthy age-matched human volunteers were also employed inthe study. Motor vehicle accidents (n=43) accounted for 65% of thecases, assaults (n=19) 29%, and falls (n=4) 6% of the cases. Braininjury was the sole medical problem in 59 of the patients, whileassociated skull fractures were present in seven cases. Criteria forexclusion included: 1. severe renal damage; 2. multiple peripheralinjuries; 3. cardiopulmonary problems; 4. hypertension; and 5. diabetes.In addition, patients on low dietary magnesium intake and on certaindrugs causing hypomagnesemia such as diuretics, antibiotics, digitalis,etc. were excluded. Patients with known histories of alcohol abuse (n=6)and drug abuse (i.e., cocaine, n=2) were included because they were ofinterest. Three patients had blood alcohol levels >200 mg/dl.

Bloods were drawn by venipuncture for routine serum laboratorychemistries (e.g. electrolytes, glucose, BUN, blood gases, creatinine)in most grade II subjects and all grade II subjects as well as healthycontrol subjects and processed by automated analyzers. In addition,blood was drawn (anaerobically) for IMg²⁺, TMg and ICa²⁺ by venipunctureusing standard red-top Vacutainer tubes. The latter was centrifuged(3000-4000 pm) for 10-15 min after clotting and processed with a novelion selective electrode (ISE) for IMg²⁺ using a NOVA Biomedical SlatProfile 8 Analyzer which can yield measurements within 2 min. Total Mgwas measured by atomic absorption spectrophotometry using a Perkin-ElmerModel Zeeman 500 and a Kodak Ektachem DT-60. The mean values usingeither technique were identical. In solution, sera of these patientswere also processed for levels of ionized calcium (ICa²⁺), sodium,potassium and hydrogen ions using selective electrodes. In order tomaintain normal pH, precautions were taken to maintain the samplesanaerobically. Precautions were also taken to prevent hemolysis, andmost blood samples were analyzed the same day. In some cases, the latterwas not possible, and in these cases the fresh anaerobically-drawn serawere frozen at -10° C. and were analyzed the next day.

Fifteen patients were administered IV fluids (<1000 ml) when blood wasdrawn. Similar control infusions were given to some patients in order todetermine if this degree of hemodilution, per se, had any significantinfluence on the observed ionized hypomagnesemia.

All patients underwent complete neurological examinations and CT scans,except a few (n=3) without loss of consciousness (LOC) who did not haveCT scans. The patients were divided into three groups and gradedaccording to severity of HT: grade I (n=8) had no LOC; grade II (n=52)had concussions, sudden brief traumatic disturbance of brain functionincluding LOC but without demonstrable anatomic lesion of brain on CTscan; and grade III (n=6) had sudden traumatic disturbance of brainfunction associated with identifiable CT lesion of brain tissue.

Mean values were calculated for serum IMg²⁺, total Mg (TMg), ICa²⁺,ICA²⁺ /IMg²⁺ and percent ionized Mg (IMg²⁺ /TMg×100). Mean values±S.E.M.were compared for statistical significance using Students "t" test,paired t test ANOVA with Scheffes' contrast test, where appropriate.Correlation coefficients, where appropriate, were also calculated usingthe method of least squares. A "P" value <0.05 was consideredsignificant.

The studies showed that acute head trauma is associated with earlydeficits in IMg²⁺, which are related to the severity of the injury(Table 8). However, TMg values were not significantly different betweengrade I, grade II or grade II HT, when compared to normal, healthy humansubjects or patient controls (data not shown, identical to healthy,human subjects). Severe head trauma (grade III) resulted in significantdepression of IMg²⁺. The ionized fraction of magnesium in serum of headtrauma patients demonstrate a progressive loss consonant with theseventy of the head trauma (Table 8). Administration of IV fluids (1000ml) did not significantly alter either the IMg²⁺ or % IMg²⁺ in the HTpatients.

                                      TABLE 8                                     __________________________________________________________________________    Serum Ionized, Total and Percent Ionized Magnesium After Head Trauma                          DEGREE OF HEAD TRAUMA                                         PARAMETERS                                                                             CONTROLS                                                                             GRADE I GRADE II                                                                              GRADE III                                     __________________________________________________________________________    IMg.sup.2+  (mM/L)*                                                                    0.585 ± 0.005                                                                     0.55 ± 0.007**                                                                     0.49 ± 0.014**                                                                     0.44 ± 0.03**                                       (60)*  (8)     (52)    (6)                                           TMg (mM/L)                                                                             0.81 ± 0.008                                                                      0.823 ± 0.015                                                                      0.792 ± 0.020                                                                      0.73 ± 0.04                                         (60)   (8)     (52)    (6)                                           % IMg.sup.2+                                                                           71.6 ± 0.58                                                                       66.8 ± 1.13**                                                                      63.1 ± 1.78++                                                                      60.0 ± 1.00**                                       (60)   (8)     (52)    (6)                                           __________________________________________________________________________     *IMg.sup.2+  = ionized magnesium; TMg = total magnesium; % IMg.sup.2+  =      ionized serum Mg                                                              +Number of subjects. Patients control values were virtually identical to      healthy volunteers.                                                           **Significantly different from controls and all other values (P < 0.01).      ++Significantly different from controls (P 0.01).                             ***Significantly different from controls and grade I HT (P < 0.01).      

Comparison of the subgroups of head injury patients showed no differencebetween IMg²⁺ levels in motor vehicle accidents (MVA's), assaults, orfalls (Table 9) but all mean values were significantly depressedcompared to controls. Although mean values for TMg in these three typesof etiologies varied from controls there were no differences in TMgbetween these patients. However, there were significant differences in %IMg²⁺ between the three types of initiating circumstances, i.e., fallsproduced the greatest deficit in % IMg²⁺ with MVA's producing the least.

                                      TABLE 9                                     __________________________________________________________________________    Serum Ionized, Total and Percent Ionized Magnesium After Head Trauma          Caused by Motor Vehicle Accidents, Assaults and Falls                                         Etiology of HT                                                PARAMETERS                                                                             CONTROLS                                                                             MVA     ASSAULTS                                                                              FALLS                                         __________________________________________________________________________    IMg.sup.2+  (mM/L)                                                                     0.585 ± 0.005                                                                     0.53 ± 0.007+                                                                      0.53 ± 0.018+                                                                      0.545 ± 0.011+                                      (60)+* (43)    (19)    (4)                                           TMg (mM/L)                                                                             0.81 ± 0.008                                                                      0.79 ± 0.012                                                                       0.827 ± 0.032                                                                      0.92 ± 0.062                                        (60)   (43)    (19)    (4)                                           % IMg.sup.2+                                                                           71.6 ± 0.58                                                                       66.9 ± 0.91+                                                                       64.1 ± 1.73+                                                                       59.0 ± 2.59**                                       (60)   (43)    (19)    (4)                                           __________________________________________________________________________     *Number of subjects                                                           +Significantly different from controls (P < 0.01)                             **Significantly different from controls and MVA (P < 0.01)               

Acute head trauma was associated with early deficits in ICa²⁺, which wasrelated to the severity of the injury (Table 10). Very severe headtrauma (grade III) resulted in almost a 20% depression of ICa²⁺, andthere was a significant increase in the relative amount of ICa²⁺ toIMg²⁺. However, none of the other serum analytes measured, includingsodium and potassium, or hydrogen ions demonstrated any abnormalities.

                  TABLE 10                                                        ______________________________________                                        Serum Ionized Calcium And Ionized Calcium to Magnesium                        Ratios After Head Trauma                                                      DEGREE OF HEAD  ICa.sup.2+                                                    TRAUMA          (mM/L)      ICa.sup.2+ /Mg.sup.2+                             ______________________________________                                        Controls        1.19 ± 0.015                                                                           2.05 ± 0.053                                                   (60)        (60)                                              Grade I         1.06 ± 0.015*                                                                          1.93 ± 0.11                                                    (8)         (8)                                               Grade II        1.01 ± 0.02*                                                                           2.06 ± 0.08                                                    (52)        (52)                                              Grade III       0.96 ± 0.02+                                                                           2.23 ± 0.034+                                                  (6)         (6)                                               ______________________________________                                         ICa.sup.2+  = ionized calcium                                                 () = number of subjects                                                       *Significantly different from controls (P < 0.001)                            +Significantly different from controls and grade I head trauma. (P < 0.01                                                                              

Although patients with isolated skull fractures all exhibitedsignificant deficits in serum IMg²⁺ and ICa²⁺ when compared to patientswith grade I head trauma, these values were not lower than those seenfor grade II HT and were not as low as seen in grade III HT (Table 11).

                  TABLE 11                                                        ______________________________________                                        Serum Ionized Magnesium, Ionized Calcium, Total and Percent                   Ionized Magnesium in Acute Head Trauma Patients with Isolated                 Skull Fractures                                                               IMg.sup.2+                                                                              ICa.sup.2+                                                          (mM/L)    (mM/L)       TMg       % IMg.sup.2+                                 ______________________________________                                        0.51 ± 0.02*                                                                         0.98 ± 0.043**                                                                          0.79 ± 0.07                                                                          64.6 ± 1.36+                              ______________________________________                                         n = 5 subjects                                                                *Significantly different from controls and grade I HT (Table 8, P < 0.02)     **Significantly different from controls and grade I HT (Table 10, P <         0.05)                                                                         +Significantly different from controls and grade III HT (Table 8, P <         0.02)                                                                    

Patients with histories of alcohol abuse or drunk on arrival in theemergency room (blood alcohol >200 mg/dl) showed significant deficits ofIMg²⁺ and ICa²⁺ when compared with patients with cocaine abuse orcontrol groups (Table 12). In addition, there was a significant increasein the relative amount of ICa²⁺ compared to IMg²⁺.

                  TABLE 12                                                        ______________________________________                                        Serum Ionized Magnesium, Ionized Calcium, Total and Percent                   Ionized Magnesium in Acute Head Trauma Patients with                          Alcohol Abuse                                                                 IMg.sup.2+                                                                             ICa.sup.2+  TMg             Ca.sup.2+ /                              (mM/L)   (mM/L)      (mM/L)   % IMg.sup.2+                                                                         mg.sup.2+                                ______________________________________                                        0.48 ± 0.029*                                                                       1.05 ± 0.044**                                                                         0.748 ±                                                                             64.7 ± 2.53                                                                       2.22 ±                                                     0.059           0.12**                                   ______________________________________                                         n = 6 subjects                                                                *Significantly different from controls and grade I HT (Table 8, P < 0.01)     **Significantly different from controls (Table 10, P < 0.01)             

The findings provide the first evidence for divalent cation changes inblood early after traumatic brain injury which are of diagnostic valuein the assessment of the severity of head injury, making estimations ofprognosis in such patients more reliable. The method of analyzingionized magnesium and ionized calcium can be used to monitor theresponse of HT to therapeutic intervention. In addition, the findingssupport early intervention with Mg salts after traumatic brain injury.

EXAMPLE XVII DIAGNOSIS & PROGNOSIS IN HYPERTENSION

Applicants hypothesized that many hypertensive human subjects might beexpected to exhibit reduction in ionized Mg²⁺ (Altura, B. M. et al.Federation Proc. 40:2672-2679, 1981; Altura, B. M. et al. Science223:1315-1317, 1984) and that treatment of such hypertensive subjectswould restore ionized Mg²⁺ to normal. Therapy of such patients should besignified by adjustments of plasma ionized Mg²⁺ and would be a valuableadjunct for diagnosis and treatment of such patients. Data on more than30 normotensive (0.52-0.67 mM), untreated hypertensive (0.42-0.60) andtreated hypertensives (0.56-0.63), using the methodology of the presentinvention, supported this idea.

Ionized magnesium, serum total magnesium, and plasma renin activity(PRA) in fasting normotensive (NT) (n=20), essential hypertensive (n=28)subjects was monitored before, and 60, 90, 120 and 180 minutes afteroral glucose loading (100 gm). Results were compared to intracellularfree magnesium values obtained at the same time intervals as measuredusing ³¹ P-NMR spectroscopy.

The average ionized Mg²⁺ values in fasting normotensive subjects were0.63±0.01 mM. Ionized Mg²⁺ values in essential hypertensives, as agroup, was 0.60±0.01 mM. Ionized Mg²⁺ values was significantly lower foressential hypertensives who had high plasma renin activity (0.57±0.01mM,sig=0.05 vs NT) compared to normotensives or essential hypertensives whohad low plasma renin activity (0.62±0.01 mM, sig=NS vs NT).

The ionized Mg²⁺ values for non-insulin dependent diabetics wasconsistently lower than for normotensives (0.57±0.01mM, p<0.05 vs NT).For all subjects, fasting ionized Mg²⁺ was related to Mgi (r=0.62,p<0.01).

Oral glucose loading reciprocally lowered intracellular Mg (219±12 to193±13 μM, p<0.01), while elevating ionized Mg²⁺ levels (0.60±0.02 to0.64±0.02 mM, p<0.01). Lastly, the dynamic changes in intracellular Mgand circulating ionized Mg²⁺ were also correlated (r=0.612, p<0.05).Total magnesium values did not differ in non-insulin dependentdiabetics, or after glucose loading.

These dam demonstrate that non-insulin dependent diabetics and essentialhypertensives with high plasma renin activity have significantly lowercirculating Mg²⁺ than normotensives subjects.

The dam also demonstrates that them is physiological transport ofcellular ionic magnesium into the extracellular space in response tooral glucose loading.

Monitoring ionized Mg²⁺ concentrations in humans or animals, by thepresent invention, now makes it possible to diagnose, prognoses andtreat hypertensive subjects.

EXAMPLE XVIII DIAGNOSIS AND PROGNOSIS OF PATIENTS WITH IDIOPATHICINTRACRANIAL HYPERTENSION

Idiopathic intracranial hypertension (IIH), is a well-defined syndromeof unknown etiology characterized by increased intracranial pressure(ICP), papilledema, normal intracranial anatomy and normal cerebrospinalfluid (CSP) which typically affects young obese women [Ahlskog, J. E. etal. Ann. Intern. Med. 97:249-256, 1982]. IIH has been described inassociation with diverse contributing factors such as certain diseasestates, endocrinologic abnormalities, ingestion of certain exogenousagents as well as in pregnancy and steroid withdrawal [Aslokog, J E etal. Ann. Intern. Med. 97:249-256, 1982; Donaldson, J. O. Neurology 31:877-880, 1981; Corbett, J. J. Can. J. Neurol. Sci. 10:22-229; 1983;Johnston, I. et al. Arch. Neurol. 8:740-747, 1991; Couban et al. Can.Med. Assn. J. 145: 657-659, 1991]. The cerebrospinal fluid in IIH ischaracterized as being under increased pressure, acellular incomposition with normal glucose content, and normal to low-normalcontent. Some abnormalities have been observed in CSF in IIH, e.g.,certain hormone levels appear to be elevated (i.e., vasopressin,estrone), whereas estrogen levels are often depressed [Seckl, J. et al.J. Neurol. Neurol. Neurosurg. Psychiatry 51:1538-1541, 1988; Donaldson,J. O. et al. J. Neurol. Neurosurg. Psychiatry 45:734-736, 1982; Srenson,P. S. et al. Arch. Neurol. 43:902-906, 1986]. Many investigators believethat a single underlying mechanism may be responsible for the increasein ICP. It has been suggested that IIH is a syndrome of varying braincompliance and that a vascular mechanism may have an importantpathogenetic role [Johnston, I. et al. Arch. Neurol. 48:740-747, 1991;Quincke, H. Dtsch. Z. Nerv. 9:140-168, 1907; Felton, W. L. et al.Neurol. 41 (Suppl.): 348 (Abstr.), 1991.] It is now possible, by thepresent invention, to diagnose, prognoses, and treat IIH, by monitoringthe levels of total Mg, IMg²⁺ and ionized Ca²⁺ (ICa²⁺) in the serum andCSF of patients with IIH.

Patients with IIH were identified on admission to the hospital. Allpatients fulfilled the modified Dandy criteria for the diagnosis of IIH.All patients were obese young women. Four of five patients (subject nos.2-5) had no history of any possible contributing factor other thanobesity. One subject (subject no. 1) had a history of post-partumsagittal sinus thrombosis two years prior to entering the study. Sevencerebral spinal fluid (CSF) specimens were obtained from the fivesubjects after informed consent. Single specimens were obtained fromsubject numbers 1, 3 and 4, and two specimens each (at different timesduring attack) from subjects 2 and 5. Serum was obtained anaerobicallyafter venipuncture from subjects 2, 3 and 5 during acute symptomaticexacerbations of IIH. Normal, healthy faculty and students volunteeredto serve as controls.

Anaerobically-maintained serum and CSF were used to measure levels ofIMg²⁺, ICa²⁺, sodium, potassium and H+ (pH) by ion selective electrodes.Total Mg in CSF and serum were determine by atomic absorptionspectroscopy and a Kodak DT-60 Ektachem Analyzer. Percent IMg²⁺ wascalculated for both the CSF and serum samples. Mean values±S.E.M. werecalculated and compared for statistical significance by a non-pairedStudent's t-test. A p-value less than 0.05 was considered significant.

The CSF levels of total Mg (TMg) were normal in all patients (Table 13).CSF levels of IMg²⁺ (0.98±0.046 mM/L), ICa²⁺ (0.89±0.025 mM/L) and %IMg²⁺ (80.4±4.49) were considerably below the normal ranges in patientswith idiopathic intracranial hypertension:, CSF ionized Na+ and K+ aswell as pH were normal in IIH.

                  TABLE 13                                                        ______________________________________                                        Ionized Mg.sup.2+, Ionized CA.sup.++, Total Mg and Percent Ionized            Mg in Cerebral Spinal Fluid of Patients Diagnosed as Having                   Pseudotumor Cerebri                                                           Subject    mM/L                                                               Number     IMg.sup.2+                                                                             ICa.sup.2+                                                                             TMg    % IMg.sup.2+                              ______________________________________                                        1          0.94     0.79     1.15   81.7                                      2          1.21     0.87     1.19   100.0                                                0.98     0.93     1.23   79.6                                      3          0.89     0.89     1.32   67.4                                      4          0.82     0.84     0.90   91.1                                      5          1.01     0.98     1.48   68.2                                                 1.02     0.96     1.36   75.0                                      Mean ± S.E.M.                                                                         0.98 ±                                                                              0.89 ±                                                                              1.23 ±                                                                            80.4 ± 4.49*                                      0.046*   0.025*   0.069                                            Normal Range                                                                             1.12-1.19                                                                              1.10-1.23                                                                              1.15-1.35                                                                            90-98                                     ______________________________________                                         *Significantly different normal values (P < 0.01).                       

Serum levels of TMg, IMg²⁺, Ca²⁺, Na⁺, K⁺ and H⁺ in IIH did not differfrom normal, healthy subjects (Table 14). Our findings are compatiblewith the idea that ionic aberrations and alterations in vascular tone inthe arachnoid granulations or permeability of the vascular walls mayhave important pathogenetic roles in pseudotumor cerebri. Interventionwith Mg could have ameliorative actions in patients with IIH.

                  TABLE 14                                                        ______________________________________                                        Ionized Mg.sup.2+, Ionized Ca.sup.2+, Total Mg and Percent Ionized Mg         in Serum of Patients with IIH Compared to Normal,                             Healthy Subjects                                                                      Mean Values (mM/L ± S.E.M.)                                        Group   N     IMg.sup.2+                                                                             ICa.sup.2+                                                                             TMg    % IMg.sup.2+                           ______________________________________                                        IIH     3     0.60 ±                                                                              1.10 ± 0.115                                                                        0.89 ±                                                                            67.6 ± 3.98                                       0.034             0.013                                         Controls                                                                              23    0.58 ±                                                                              1.15 ± 0.024                                                                        0.85 ±                                                                            68.2 ± 0.84                                       0.006             0.022                                         ______________________________________                                    

EXAMPLE XIX DIAGNOSIS & PROGNOSIS IN PREGNANCY-INDUCED PRE-ECLAMPSIA,HYPERTENSION, CONVULSIONS AND FETAL GROWTH RETARDATION

Using the present method of sample processing and an ISE for measuringionized Mg²⁺, a study was undertaken to determine ionized and total Mglevels in umbilical venous and arterial blood in normal and abnormalpregnant patients. Correlations were made between Mg²⁺ levels andmaternal and neonatal pathological states. The study consisted of 64pregnant patients of which 38 had no maternal or neonatal complicationsand 26 had one or more or the following abnormalities: toxemia,transient hypertension, gestational diabetes, premature labor during thecurrent pregnancy or delivery prior to 38 weeks, growth retardednewborn, chorioamnionitis, or ABO incompatibility in the newborn. AllAPGAR scores were 9,10. There were no differences between the groupswith regard to maternal age, race, parity, percentage of indigentpatients, mode of delivery, epidural analgesis, use of Pitocin, use ofoxygen in labor, ECA at delivery, mean birth weight or sex of newborn.Three patients in the abnormal group received Mg therapy for toxemia andhad venous samples taken. The results are expressed as mean (mM)±SEM. Innormal pregnancies, the mean umbilical venous plasma (or serum) ionizedMg²⁺ level was 0.51±0.01 (N=38), which approximates the lower end of thenormal range values in the venous plasma of non-pregnant women. The meanumbilical arterial ionized Mg²⁺ level in normal pregnancies was0.48±0.01 (N≦24), demonstrating significant differences in the amount ofionized Mg²⁺ which enters or leaves the fetus.

Pregnant women who had one or more of the various maternal pathologicalconditions listed above had a significantly lower mean venous plasma orserum ionized Mg²⁺ level (0.44±0.014) than the normal group. Thesubgroup of pregnant women who exhibited transient hypertension had amean venous ionized Mg²⁺ level of 0.43±0.015, which is almost a 20%deficit compared to the normal group. Two patients that hadchorioamnionitis had the lowest values of umbilical venous Mg²⁺ in theentire study population, 0.25 mM and 0.38 mM. The percent of the totalMg which was ionized was 67.3±1.89 in the normal pregnancies and64.5±2.31 in the abnormal ones, an insignificant difference. One of thepatients with chorioamnionitis had only 49% ionized Mg²⁺, however.

The study population consisted of mothers in labor, of which 51 wererandomly selected, after a chart review revealed that they had nocomplications prior to labor and were greater than 37 weeks gestationalage. Thirteen of these patients developed transient hypertension inlabor and were evaluated separately. The remaining 38 patients comprisedthe normal study group. Venous blood samples from a separate group of 26normal mothers admitted to the Labor and Delivery Suite in labor or forelective Cesarean section, and 42 samples from another group ofnon-pregnant healthy women, ages 19 through 45, were evaluated forcomparison purposes.

Transient hypertension in labor was defined as repeated systolic bloodpressures of ≧140 mmHg in the first stage of labor in patients withoutproteinuria or other signs of symptoms of preeclampsia and with no othercomplications in pregnancy. Blood pressures were measured in betweencontractions with the women in a semirecumbant position and the arm atheart level. Blood pressures of patients with this diagnosis were normalpost-partum.

Newborns were considered large for gestational age (LGA) or small forgestational age (SGA) if they were greater than the 90th percentile orless than the 10th percentile in weight according to the homograms ofBatagglia et al. (J. Pediatr. 1967, 71:159-163). If any study neonatewas admitted to the high risk nursery, the reason for the admission wasto be documented. Patients in Labor and Delivery were not allowed to eator drink anything and were given intravenous Ringers-lactate solution(Baxter Health Care Corp., Deerfield, Ill.) at 125 ml. per hour.Occasional patients received meperidine HCI (50 mg IV, Elkins Sinn,Inc., Cherry Hill, N.J.) and promethazine HCI (25 rag. IV, Elkins Sinn)for analgesia, as well as an oxytocin (Parke Davis, Morris Plains, N.J.)infusion (1-15 mU/min) for labor augmentation. When epidural analgesiaor anesthesia was given, a test dose consisting of 3 ml. of 1.5%lidocaine HCI with epinephrine (1:200,000 dilution, Astra PharmaceuticalProds., Westborough, Mass.) followed by a first dose containing the samemedication was given. This was followed by a continuous infusion ofbupivicaine HCI (0.083%, Astra Pharm.) and fentanyl (2 μg/ml, ElkinsSinn, Inc., Cherry Hill, N.J.). Patients who had epidural anesthesia forCesarean sections were premedicated with a scopolamine patch (1.5 mg,CIBA-Geigy, Summit, N.J.) and a sodium citrate and citric acid oralsolution (30 ml. Willen Drug Co., Baltimore, Md.) orally. They thenreceived 13 to 20 ml. of 2% lidocaine and epinephrine (1:200,000, AstraPharm.) or 15 to 20 ml. of chloroprocaine (3%, Astra Pharm.) to achievean anesthetic block at the T4 level. Prior to epidural analgesia inlabor, or epidural anesthesia for Cesarean procedures, all patientsreceived an intravenous infusion of 1000 ml or 1500 ml, respectively, ofRingers-lactate solution over 15 minutes for prehydration.

Assessment of Serum Ionized Magnesium, Total Magnesium and IonizedCalcium

A 7 to 20 cm section of umbilical cord was double-clamped at deliveryand samples of venous and arterial blood were aspirated separately,placed in plain red-stoppered Vacutainer tubes and allowed to clot atroom temperature. Peripheral blood samples were also obtained in anage-matched group of normal, healthy non-pregnant women by venipuncturevia Vacutainer tubes under anaerobic conditions. All blood samples werethen centrifuged at 3000 g for 15 minutes under anaerobic conditions,and an aliquot of serum was removed under anaerobic conditions andstored at -20° C. Samples were analyzed within two weeks using a NOVABiomedical Slat Profile 8 Analyzer (NOVA Biomedical Corp., Waltham,Mass.) to measure IMg²⁺ and ICa²⁺. In addition, TMg levels were measuredwith either atomic absorption spectroscopy (AAS) or a Kodak EctachemDT-60 Analyzer (12). There was no significant differences formeasurement of TMg between AAS or the Kodak Analyzer.

Mean levels±S.E.M.) of the cations and of the fractions of IMg²⁺ werecalculated for each patient and control group. An attempt was made tosee if there were any correlations between the levels determined andclinical parameters and if there were any relationships between thecations. The numbers of samples within each subgroup varied due tosample loss. Statistical analyses were performed using the SPSSstatistical package (Notusis, M. J. SPSS/PCAT Release 3.1, Chicago, SPSSInc., 1986) for the unpaired Student's t test, Chi-square analysis, andPearson's correlation coefficient. All results are expressed asmeans±standard error of the mean (SEM) unless otherwise indicated. A pvalue of less than 0.05 was considered significant.

Ranges of IMg²⁺, Total Mg, % IMg²⁺ and ICa²⁺

The IMg²⁺ levels for both umbilical vein and artery, and the maternalvein, of the pregnant subjects remained within narrow ranges, i.e.0.49-0.53 mmol/l, 95% confidence interval for the umbilical vein;0.46-0.53 mmol/l, 95% confidence interval for the umbilical artery; and0.46-0.51, 95% confidence intervals for the maternal venous blood. Innon-pregnant women the serum range was 0.55-0.67 mmol/L. The ranges fortotal Mg were 0.70-0.745 mmol/l for the umbilical vein, 0.70-0.78 mmol/lfor the umbilical artery, and 0.74-0.82 mmol/l for maternal blood. Innon-pregnant women, the serum range was 0.70-0.98 mmol/L. The ranges forthe ionized fractions were 68.6-72.2% for the umbilical vein, 61.5-67.8%for the umbilical artery, and 60.5-65.1% for maternal venous blood. Innon-pregnant women, the serum % IMg²⁺ ranges from 67.2-75.2%.

The ICa²⁺ levels (95% confidence intervals) for both umbilical vein andartery had much wider ranges than those for IMg²⁺, i.e., 0.97-1.09mmol/l for the artery and 1.11-1.23 mmol/l for the vein. For maternalvenous blood, the ICa²⁺ range was (also much wider than for IMg²⁺,i.e.,) 0.87-1.28 mmol/l. In non-pregnant women the peripheral serumrange was 1.10-1.30 mmol/L.

Relationships Between IMG²⁺ Levels, Fractions of Mg and Levels of ICa²⁺in Umbilical Vessels of Normal Subjects

Mean umbilical arterial IMg²⁺ remained slightly (by 0.03 mmol/l) butsignificantly (p<0.05) lower than umbilical venous IMg²⁺ in the normalpatients (Table 15), but there was a highly significant correlation(r=0.79) between both values (p<0.0001). The differences in the TMgbetween the cord blood in the two vessels were not significant, and thearterial TMg was highly correlated with the venous TMg (r=0.76)(p<0.0001). Arterial IMg²⁺ also correlated with arterial total Mg(r=0.56) (p<0.01) and similarly, venous IMg²⁺ correlated with venoustotal Mg (r=0.69) (p<0.0001).

                  TABLE 15                                                        ______________________________________                                        Ionized Mg.sup.2+, Total Mg and % IMg.sup.2+  in Umbilical Arterial and       Venous Cord Blood of Normal Subjects                                          Arterial          Venous                                                      IMg.sup.2+                                                                           TMg                IMg.sup.2+                                                                           TMg    %                                     (mmol/l)                                                                             (mmol/l) % IMg.sup.2+                                                                            (mmol/l)                                                                             (mmol/l)                                                                             IMg.sup.2+                            ______________________________________                                        0.49 ±                                                                            0.76 ±                                                                              63.5 ± 1.87                                                                          0.51 ±                                                                            0.72 ±                                                                            70.2 ±                             0.01   0.02     (22)      0.001**                                                                              0.01   0.94***                               (24)*  (23)               (38)   (38)                                         ______________________________________                                         Values are means ± S.E.M.                                                  *Represents number of different subjects.                                     **Significantly different from arterial IMg.sup.2+  (p < 0.05)                ***Significantly different from arterial % IMg.sup.2+  (p < 0.01)?       

The ionized fraction of Mg was significantly lower in the umbilicalarteries than in the veins (p<0.01) and in both vessels the fractionswere negatively correlated with the TMg levels (r=-0.42, p<0.05 inarteries and r=0.54, p<0.001 in the veins). In other terms, as the TMgrises, the % IMg²⁺ falls.

The mean ICa²⁺ level in the umbilical vein (1.20±0.02 mmol/L) wassignificantly higher than in the umbilical artery (1.03±0.03 mmol/L)(p<0.001). The umbilical arterial ICa²⁺ level was significantlycorrelated to the IMg²⁺ level (r=0.44, p=0.02), but this type ofcorrelation was not seen in the vein. On the other hand, the ICa²⁺levels correlated with the Mg fractions in both the umbilical artery(r=0.28, p<0.01) and umbilical vein (r=0.28, p<0.01) and umbilical vein(r=0.532, p<0.05).

Comparison of Relationships Between IMg²⁺ Levels, Fractions of Mg andLevels of IC²⁺ in Peripheral Venous Blood Samples of Normal Pregnant andNon-pregnant Women with Samples from Umbilical Vessels

The mean level of IMg²⁺ in the peripheral venous blood of normal,pregnant women (0.485±0.01, n=26) was slightly but significantly lessthan that in the umbilical vein (Table 15) (p<0.05) and similar to thatin the umbilical artery. The mean TMg in the peripheral blood of themother (0.78±0.02, n=26) was significantly higher than in the umbilicalvein (p<0.001), and the ionized fraction of Mg in the mother (62.2% )was significantly less than in the umbilical vein (p<0.001), and theionized fraction of Mg in the mother (62% ) was significantly less thanin the umbilical vein (p<0.001 ), but similar to that in the artery. Inthe maternal veins, as in the umbilical vessels, the IMg²⁺ levelscorrelated with the total Mg levels (r=0.44, p<0.01 for both), and theIMg²⁺ ionized fraction was negatively correlated with the total Mg(r=-0.48, p<0.01).

As in the umbilical arteries, maternal venous ICa²⁺ (1.09±0.01 mmol/l)positively correlated (r values=0.5-0.6, p<0.002) with both maternalvenous IMg²⁺ levels and with Mg fractions (p<0.01 for both). Also, aswith umbilical arterial blood, this maternal peripheral venous ICa²⁺level was significantly less than the umbilical venous level (p<0.001).In contrast to these values, the peripheral venous IMg²⁺ ofnon-pregnant, age-matched healthy women is approximately 15-20% higher(0.60±0.0005 mmol/L, n=42) than that of umbilical cord blood or maternalvenous blood of women in labor. The peripheral venous TMg and % IMg²⁺ inthe non-pregnant age-matched healthy women are 0.83±0.06 mmol/L and71.6±0.58%, respectively.

Correlation of Demographic Variables with IMg²⁺ and ICa²⁺ Levels andFractions in Umbilical Vessels of Normal Patients

In the normal study group, the maternal age ranged from 19 to 40 years,gestational age from 37.5 to 42.0 weeks, and birthweight from 2,608 to4,706 grams. Within those ranges, there were no significant correlationsbetween umbilical arterial or venous levels of IMg²⁺, TMg or ionizedfraction of Mg²⁺ and the maternal or gestational ages and birthweights(Pearson's correlation coefficient p>0.05). However, there was apositive correlation between umbilical arterial ICa²⁺ and birthweight(r=0.36, p=0.04).

Multiparous patients had approximately an 8% lower mean umbilical venousIMg²⁺ than primiparous patients, (0.49±0.01 [n=24], versus 0.53±0.02mmol/l [n=13] p=0.02. Differences in TMg levels or fractions were notsignificantly different nor were there differences in the arterialsamples.

There were no differences between clinic and private patients in meanIMg²⁺, TMg levels, % IMg²⁺ or ICa²⁺ levels.

With regard to race, Blacks and Hispanics had similar levels ofumbilical arterial and venous IMg²⁺ compared to Whites, but Asians hadapproximately 8% and 27% higher mean venous and arterial IMg²⁺ levelswhen compared to Whites (0.54±0.02 mmol/L versus 0.50±0.01 mmol/Lvenous, and 0.57±0.02 versus 0.45±0.01 mmol/L arterial, p=0.01 and0.001, respectively). The percent ionized Mg²⁺ fractions, however, didnot differ between Asians and Whites. On the other hand the percentionized Mg²⁺ was significantly higher in Blacks than Whites in theumbilical vein (73.5±1.3 versus 68.8±1.1, p=0.03). In Hispanics, themean TMg level in the umbilical veins was significantly lower than thatof Whites (0.66±0.02 versus 0.73±0.02, p=0.04).

ICa²⁺ levels also showed some variation with race. Asians appeared tohave the highest mean umbilical venous ICa²⁺, compared to Whites(1.27±0.04 versus 1.19±0.03 mmol/L) but this difference did not achievestatistical significance. The umbilical arterial 1Ca²⁺ of Hispanics wasalmost significantly higher than that of Whites (1.12±0.04 versus0.99±0.05 mmol/L, p=0.06).

Influence of mode of Delivery and Medications on 1 Mg²⁺ Levels, MgFractions and 1Ca²⁺ Levels

As seen in Table 16, there were no significant differences in arterialor venous IMg²⁺, TMg and % IMg²⁺ levels in patient delivered by Cesareansection compared to those who had spontaneous vaginal delivery. Butpatients with operative vaginal delivery had both increased arterial andvenous IMg²⁺ (p<0.05)and venous TMg (p=0.04) compared to patients withspontaneous vaginal delivery. ICa²⁺ levels did not vary with mode ofdelivery.

                                      TABLE 16                                    __________________________________________________________________________    Ionized Magnesium Levels and Fractions and Ionized Ca2+ Levels in             Umbilical                                                                     Arteries and Veins of Normal Patients Versus Mode of Delivery and             Anesthesia                                                                                IMg.sup.2+                                                                           Total Mg                                                                             % Ionized                                                                           ICa.sup.2+                                    __________________________________________________________________________    Mode of Delivery:                                                             NSVD      v 0.51 ± 0.01                                                                       0.72 ± 0.02                                                                       69.8 ± 1.1                                                                       1.22 ± 0.02                                            (21)   (21)   (21)  (21)                                                    a 0.48 ± 0.01                                                                       0.75 ± 0.03                                                                       63.9 ± 2.3                                                                       1.06 ± 0.04*                                           (12)   (11)   (11)  (12)                                          Vaginal   v 0.55 ± 0.22**                                                                     0.80 ± 0.04**                                                                     68.8 ± 2.7                                                                       1.21 ± 0.09                                Operative   (5)    (5)    (5)   (5)                                                     a 0.57 ± 0.03**                                                                     0.82 ± 0.04                                                                       69.7 ± 0.38                                                                      1.10 ± 0.07                                            (3)    (3)    (3)   (3)                                           Cersarean v 0.49 ± 0.01                                                                       0.69 ± 0.02                                                                       72.0 ± 1.6                                                                       1.17 ± 0.03                                            (11)   (11)   (11)  (11)                                                    a 0.45 ± 0.02                                                                       0.71 ± 0.02                                                                       63.9 ± 2.3                                                                       0.98 ± 0.05                                            (18)   (17)   (16)  (18)                                          Use of Epidural:                                                              yes       v 0.50 ± 0.01                                                                       0.70 ± 0.02                                                                       70.9 ± 1.1                                                                       1.20 ± 0.02                                            (25)   (25)   (25)  (25)                                                    a 0.48 ± 0.01                                                                       0.72 ± 0.02                                                                       65.6 ± 15                                                                        1.06 ± 0.03*                                           (18)   (17)   (16)  (18)                                          no        v 0.53 ± 0.01.sup.+                                                                 0.75 ± 0.02                                                                       69.3 ± 1.3                                                                       1.20 ± 0.03                                            (12)   (12)   (12)  (12)                                                    a 0.49 ± 0.03                                                                       0.83 ± 0.05.sup.+                                                                 62.0 ± 4.1                                                                       0.96 ± 0.07*                                           (5)    (4)    (4)   (5)                                           __________________________________________________________________________     (Values are means ± SEM in mmol/l; numbers of patients in parentheses;     v = vein; a = artery                                                          *p < 0.01 compared to vein                                                    **p < 0.05 compared to level in same vessel of patients with NSVD             .sup.+ p < 0.05 compared to level in same vessel of patients who had          epidural                                                                 

Use of epidural analgesia or anesthesia was associated withsignificantly lower umbilical venous IMg²⁺ levels (p=0.02). The TMg wasdecreased in the umbilical arteries with use of epidural. ICa²⁺ levelsdid not vary significantly.

There were no differences in umbilical venous Mg levels or fractions ofICa²⁺ levels between patients who received intravenous or intramuscularmedications (e.g., meperidine, promethazine, oxytocin) versus those whodid not.

Relationship Between Normal Weight Grouping of Neonates and IMg²⁺Levels, Fractions of Mg and Levels of ICa²⁺

Only one infant in the normal study group was borderline SGA and, thus,comparison of cation levels was not possible for such infants. However,umbilical venous samples from 12 LGA infants, and umbilical arterialsamples from 11 of such subjects were compared with average forgestational age (AGA) neonates. In the venous samples, the ionizedfraction was significantly greater in the LGA infants than the AGAneonates (73.0±1.4 versus 68.8±1.1%, p<0.05). Umbilical arterial ICa²⁺was also significantly higher in the LGA group (1.10±0.04 versus0.97±0.04 mmol/L, p<0.05).

Comparison of Demographic Variables and Outcomes Among the DifferentStudy Groups

When the different study groups were compared with regard to maternal orgestational age, percent primigravida, race, or use of analgesia ormedications, no differences were found (Table 17). The increasedpercentages of indigent patients in the group with transienthypertension approached significance (p=0.06). Unlike the other groups,none of the patients in the transient hypersensitive group weredelivered by Cesarian section (Chi-square, p=0.02).

                  TABLE 17                                                        ______________________________________                                        Comparison of Demographic Data, Medication in Labor, and                      Neonatal Outcome in the Different Study Groups                                             Diagnosis                                                                               Transient                                                           Normal    Hypertension                                           ______________________________________                                        # of Patients  38          13                                                 Maternal Age, yr                                                                             27.9 ± 0.9                                                                             25.4 ± 1.4                                      Race:                                                                         White          19 (50.0)   6 (46.6)                                           Black          4 (10.5)    3 (23.1)                                           Hispanic       7 (18.4)    3 (23.1)                                           Asian          8 (21.0)    1 (7.7)                                            Primagravida   14 (36.8)   7 (53.8)                                           Indigent       7 (18.4)    6 (46.2)*                                          Mode of Delivery:                                                             NSVD           22 (57.9)   10 (76.9)                                          Vaginal Operative                                                                            5 (13.2)    3 (23.1)                                           Cesarean Section                                                                             11 (28.9)   0 (0)*                                             EGA            39.6 ± 0.21                                                                            39.9 ± 0.29                                     Epidural       26 (68.4)   11 (84.6)                                          Meperidine HCl,                                                                              2 (5.1)     0 (0)                                              promethazine HCl                                                              Oxytocin       13 (34.2)   2 (15.4)                                           Birth Weight (g)                                                                             3462.2 ± 79.1                                                                          3366.4 ± 122.4                                  LGA            13 (34.2)   3 (23.1)                                           Meconium       3 (7.9)     1 (7.7)                                            Male Newborn   16 (42.1)   4 (30.8)                                           ______________________________________                                         (Parametric Data are Means ± SEM; numbers in parenthesis are %)            *p < 0.05 compared to normal patients                                         .sup.+ p = 0.06 compared to normal patients                              

There were no significant differences between the neonatal groups withregard to birthweight, macrosomia, meconium and sec distribution. Noneof the neonates of the mothers with transient hypertension were SGA.None of the neonates had significant morbidity or entered the neonatalintensive care unit.

Comparison of IMg²⁺ Levels, Fractions of Mg and ICa²⁺ Levels BetweenNormal Subjects and Transient Hypertensives

The mean IMg²⁺ level in the umbilical veins, but not in umbilicalarteries, of transient hypertensives was significantly lower than thatin normal patients (Table 18). The TMg levels were similar in theumbilical veins and arteries of both types of patients. When the meanTMg levels, IMg²⁺ and mean % ionized fractions in the transienthypertensives in the artery were compared to the vein, only the % IMg²⁺was significantly different (P<0.01) from the normal subjects. Umbilicalarterial ICa²⁺ (1.00±0.07 mmol/l) was significantly lower (P<0.05) thanumbilical venous ICa²⁺ (1.17±0.003 mmol/l), as in the normal patients.However, both mean arterial and venous levels of ICa²⁺ did not differsignificantly from the mean levels of normal patients.

                  TABLE 18                                                        ______________________________________                                        Ionized Mg.sup.2+, Total Mg and % IMg.sup.2+  in Umbilical Arterial and       Venous Cord Blood of Pregnant Women with Transient                            Hypertension in Labor                                                         Arterial          Venous                                                      IMg.sup.2+                                                                           TMg                IMg.sup.2+                                                                           TMg    %                                     (mmol/l)                                                                             (mmol/l) % IMg.sup.2+                                                                            (mmol/l)                                                                             (mmol/l)                                                                             IMg.sup.2+                            ______________________________________                                        0.44 ±                                                                            0.85 ±                                                                              62.0 ± 1.68                                                                          0.46 ±                                                                            0.75 ±                                                                            68.9 ±                             0.02   0.13     (7)       0.01** 0.07   1.53***                               (7)*   (7)                (13)   (13)   (13)                                  ______________________________________                                         Values are means ± S.E.M.                                                  *Represents number of different subjects                                      **Significantly different from umbilical venous blood of normal women (P      0.01), (Table 15)                                                             ***Significantly different from arterial % IMg.sup.2+  (p < 0.01)        

When the arteriovenous differences in the umbilical vessels for mean Mglevels and fractions were compared, we could not find any significantdifferences between normal pregnant women in labor and those presentingwith transient hypertension in labor (Table 19).

It is known that labor induces significant elevations of serumepinephrine and norepinephrine. Deficits in [Mg²⁺ ] are known topotentiate the contractile effects of catecholamines on all types ofblood vessels, including umbilical arteries and veins. It has been shownthat Mg therapy can blunt the hypertensive action of epinephrine,norepinephrine and other pressors without altering its cardiotonicaction. However, patients who enter labor with a deficiency in ionizedMg²⁺ may not be able to blunt the hypertensive effects of rises innorepinephrine, epinephrine or other pressor agents related to stress.The end result is that the lower than normal IMg²⁺ allows more ICa²⁺ toenter, and be released from, the smooth muscle cells lining theperipheral blood vessels, resulting in decreased vascular lumen sizesand increases in peripheral vascular resistance.

                  TABLE 19                                                        ______________________________________                                        Arteriovenous Differences in Mg Levels and Fractions in Normal                Pregnant Women in Labor Compared to Those with                                Transient Hypertension in Labor                                                        Arteriovenous Differences                                                       IMg.sup.2+  TMg                                                    Group      (mmol/l)    (mmol/l)  % IMg.sup.2+                                 ______________________________________                                        Normal     -0.03 ± 0.01                                                                           0.07 ± 0.02                                                                          -9.05 ± 1.95                                         (23)*       (22)      (22)                                         Transient  -0.02 ± 0.01                                                                           0.04 ± 0.02                                                                          -6.14 ± 1.42                              Hypertension                                                                             (7)         (7)       (7)                                          ______________________________________                                         Values are means ± S.E.M.                                                  *Represents number of different subjects                                 

The overall dam indicate that serum or plasma ionized Mg²⁺ levels inpregnancy are of diagnostic value. Transient hypertension in labor isassociated with hypomagnesemia, which could account in large measure forthe increase in blood pressure. Therefore, the present methodology foruse in monitoring ionized Mg²⁺ concentrations throughout pregnancyallows the obstetrician to prevent pregnancy-induced pre-eclampsia,hypertension, convulsions and fetal growth retardation by treatment ofthe women with Mg²⁺ salts which elevate ionized Mg²⁺ when levels dropabnormally low. Furthermore, ionized Mg²⁺ levels may be a biochemicalmarker for following disease processes in pregnant women and theirresponse to treatment.

EXAMPLE XX IONIZED MAGNESIUM AND CALCIUM LEVELS IN CYCLOSPORINE TREATEDRENAL TRANSPLANT RECIPIENTS

Ionized Mg²⁺ and ionized Ca²⁺ were measured in 54 cyclosporine (CSA)treated renal transplant recipients (6 mos. to 7 yrs. post-transplant,mean CSA=192±19.3 ng/dl) and 34 age-matched control subjects using anion selective electrode.

Renal transplant recipients demonstrated pronounced deficits in meanionized Mg²⁺ (0.48±0.01 vs. control 0.61±0.06 mM/L, p<0.001). Theserecipients demonstrated slight deficits in mean total magnesium(0.77±0.015 vs. 0.84±0.017 mM/L, p<0.001) and no change in mean ionizedCa²⁺ (1.20±0.02 vs. 1.18±0.01 mM/L, p=NS).

Renal transplant recipients with plasma cholesterol <215 mg/dl andcontrol subjects did not show a correlation between cholesterol level,ionized Mg²⁺, ionized Ca²⁺ or total magnesium. Both ionized Mg²⁺ andtotal magnesium correlated positively (p<0.05) with plasma cholesterolin renal transplant recipients with plasma cholesterol levels >240mg/dl. Renal transplant recipients with high plasma cholesterol levelsalso demonstrated a strong negative correlation between cyclosporinelevel and ionized Mg²⁺ (p<0.01), i.e., patients with high cyclosporinelevels having the lowest ionized magnesium values.

Renal transplant recipients with high plasma cholesterol had strongpositive correlation between cyclosporine levels and ionized Ca²⁺/ionized Mg²⁺ ratios and a negative correlation between plasmacholesterol and ionized Ca²⁺ /ionized Mg.sup. 2+ ratios.

Therefore, using the method of the present invention, it has beenpossible to correlate cyclosporine toxicity with ionized Mg²⁺deficiencies in renal transplant recipients with hypercholesterolemia.The accelerated atherosclerosis noted in cyclosporine-treated renaltransplant recipients is related to alterations in ionized Mg²⁺ ratios.Ionized Mg²⁺, and not total Mg, appears to be the most sensitiveclinical parameter in cyclosporine-treated renal transplant recipients.Therefore, the method of the present invention in conjunction withmeasurements of plasma cholesterol is diagnostic and prognostic inpredicting development or exacerbation of atherosclerosis in renaltransplant recipients treated with cyclosporine. Therapeuticintervention with magnesium to bring the plasma levels of ionizedmagnesium to within the normal range of approximately 0.53-0.67 mMserves to lessen atherosclerosis in renal transplant recipients withhigh plasma cholesterol levels.

EXAMPLE XXI IONIZED CA²⁺ :MG²⁺ RATIOS

Since determinations of ionized Ca²⁺ have been suggested to be of valuein critical care medicine (Zaloga, G. P. et al. Crit. Care Med.15:813-816, 1987; Olinger, M. L. The Emerg. Med. Clin. N. Amer.7:795-822, 1989) and significant alterations in ionized Mg²⁺ can bemeasured using the present methods, it is reasonable to examine andutilize Ca²⁺ :Mg²⁺ ratios in the diagnosis and treatment of diseasestates where both of these cations could be expected to exhibit subtlechanges in body fluids. The data described herein, particularly forcardiac patients and such patients on cardiopulmonary bypass indicatethat the Ca²⁺ :Mg²⁺ ratios are significantly diagnostic and prognosticmarkers for hypotension, coronary vasospasm and dysrhythmias during andpost cardiac surgery. Additionally, Ca²⁺ :Mg²⁺ ratios may be diagnosticand prognostic in determining the seventy and progression of headtrauma, abnormal pregnancies, and hypotension.

Currently, it is recommended by the US National Academy of Sciences thathuman subjects consume in their diets 900-1000 mg/day of elementedcalcium (which=22.5-25 mmoles of Ca) and 350-400 mg/day of elementalmagnesium (which=14.4-16.5 mmoles of Mg). This represents molar ratios(Ca/Mg) of 1.36-1.74. All current diet supplements and dietarycomponents are based on these values. However, these values used by theUSA National Academy of Sciences are based on metabolic balance studiesof calcium and magnesium in human subjects. Such metabolic balancestudies are based on total calcium and total magnesium balances, not onthe biologically (or physiologically) active minerals, which are thetonic forms, i.e. ICa²⁺ and IMg²⁺.

The measurements on whole blood, plasma and serum levels of ICa²⁺ andIMg²⁺ by the method of the present invention, using an ion selectiveelectrode, yield mean ICa²⁺ levels of about 1.20 mM/L and IMg²⁺ levelsof about 0.60 mM/L. This is a molar ratio of approximately 2.00/1.Therefore, the old formulation based on total calcium and totalmagnesium is incorrect. Normal diets should contain a molar ratio ofapproximately 2.0/1.0 for Ca/Mg in order to maintain the proper bloodlevels of ionized Ca²⁺ and ionized Mg²⁺. Dietary supplements, vitaminand mineral supplements should thus be based on such a new ratio.

EXAMPLE XXII TREATMENT OF HYPO- AND HYPER-MAGNESEMIA STATES

Use of the present invention allows the physician, veterinarian andresearcher to scientifically monitor and treat hypo- or hypermagnesemiastates.

Candidates for treatment with Mg²⁺ or calcium and Mg²⁺ include animals,particularly mammals such as humans with coronary heart disease,congestive heart failure, hypomagnesemia, critical illnesses, lungdiseases, abnormal pregnancy, undergoing cardiopulmonary bypass, headtrauma, aminoglycoside (or other antibiotics) toxicity, chemotherapeuticdrug-induced hypomagnesemia or those in high risk categories for heartattack or stroke such as those with hypertension, diabetes, highcholesterol, or smokers and the like. Candidates for Mg²⁺ treatment orMg²⁺ and Ca²⁺ treatment also includes those with idiopathic intracranialhypertension, renal transplant recipients and non-insulin dependentdiabetics. The amount of Mg²⁺ administered will, of course depend uponthe severity of the condition being treated, the route of administrationchosen, and the dose of Mg²⁺, and ultimately will be decided by theattending physician or veterinarian. As a guide, a concentration ofMg²⁺, as used in the prior art include regimens similar to thosereported by clinicians for different disease states (Wacker, W. E. C.Magnesium and Man, 1980; Iseri, C. T. et al. West J. Med. 138:823-828,1983; Zaloga, G. P. In: Problems in Critical Care Vol 4, 1990; Rudnick,M. et al. APMIS 98:1123-1127, 1990; Rasmussen, H. S. et al. Lancet1:234-236, 1986; Berkelhammer, C. et al. Canadian Med. Assoc. J.312:360-368, 1985; Cohen, L. et al. Magnesium 3:159-163, 1984; Dyckner,T. et al. Brit. Med. J. 286:1847-1849, 1983; Olinger, M. L. The Emerg.Med. Clin. N. Amer. 7:795-822, 1989; Kobrin, S. M. et al. Sem. inNephrol. 10:525-535, 1990). Use of the present methodology andassessment of ionized Mg²⁺, rapidly, will make it possible to monitor apatient's response to therapeutic regimens in a precise and carefullycontrolled manner, which was heretofore not possible.

Mg²⁺ or Mg²⁺ and Ca²⁺ may be administered by any route appropriate tothe condition being treated including intravenous (IV), intraperitoneal,intramuscular, subcutaneous, oral, nasal, and the like. Preferably, theMg²⁺ is injected IV into the blood stream of the mammal being treatedespecially in acute cases of hypomagnesemia. It will be readilyappreciated by those skilled in the art that the preferred route willvary with the condition being treated.

While it is possible for the Mg²⁺ to be administered as the pure orsubstantially pure mineral, it is preferable to present it as apharmaceutical formulation or preparation. Suitable bioavailablemagnesium salts and magnesium compounds are well known in the art asdescribed in U.S. Pat. No. 4,954,349 and U.S. Pat. No. 4,546,195,incorporated herein by reference.

The formulations for the present invention, both veterinary and forhuman use, comprise Ca²⁺, Mg²⁺ or Ca²⁺ and Mg²⁺ together with one ormore pharmaceutically acceptable carriers and optionally othertherapeutic ingredients. The carrier(s) must be "acceptable" in thesense of being compatible with the other ingredients of the formulationand not deleterious to the recipient thereof. The formulations mayconveniently be presented in unit dosage form and may be prepared by anymethod well known in the pharmaceutical art.

All methods include the step of bringing into association the activeingredient with the carrier which constitutes one or more accessoryingredients. In general, the formulations are prepared by uniformly andintimately bringing into association the active ingredient with liquidcarriers or finely divided solid carriers or both, and then, ifnecessary, shaping the product into the desired formulation.

Formulations suitable for intravenous, subcutaneous, or intraperitonealadministration conveniently comprise sterile aqueous solutions of theactive ingredient with solutions which are preferably isotonic with theblood of the recipient. Such formulations may be conveniently preparedby dissolving solid active ingredient in water containingphysiologically compatible substances such as sodium chloride (e.g.0.1-2.6M), Ringers solution, parenteral solution for I.V. or oraladministration and the like, and having a buffered pH compatible withphysiological conditions to produce an aqueous solution, and renderingsaid solution sterile. These may be presented in unit or multi-dosecontainers, for example, sealed ampoules or vials.

The formulations of the present invention may incorporate a stabilizer.Illustrative stabilizers are polyethylene glycol, proteins,saccharities, amino acids, inorganic acids, and organic acids which maybe used either on their own or as admixtures. These stabilizers arepreferably incorporated in an amount of 0.11-10,000 parts by weight perpart by weight of mineral. If two or more stabilizers are to be usedtheir total amount is preferably within the range specified above. Thesestabilizers are used in aqueous solutions at the appropriateconcentration and pH. The specific osmotic pressure of such aqueoussolutions is generally in the range of 0.25-0.35 osmoles, preferably inthe range of 0.29-0.32. The pH of the aqueous solution is adjusted to bewithin the range of 7.0-8.0, preferably within the range of 7.2-7.6. Informulating the therapeutic agent of the present invention,anti-adsorption agent may be used.

It is an object of the present invention to provide a mineralcomposition for the prevention or treatment of magnesium imbalances ordeficiencies or calcium/magnesium imbalances in an adult comprisingcalcium in the form of a bioavailable pharmaceutically acceptable saltthereof and magnesium in the form of a bioavailable pharmaceuticallyacceptable salt thereof, alone or in combination to achieve or maintaina molar ratio of ionized Ca⁺⁺ /ionized Mg⁺⁺ of about 2.5:1-1:1, morepreferably about 1.5:1, and most preferably about 2:1 in the blood or amolar ratio of ionized Ca²⁺ /ionized Mg²⁺ of about 0.90:1 to 1.15:1,more preferable 0.92:1 to about 1.1:1, most preferably about 1:1 incerebral spinal fluid.

It is an object of the present invention to provide a mineralcomposition for the prevention or treatment of magnesium deficiencies,magnesium imbalances and calcium/magnesium imbalances based on themeasured ionized level of Mg²⁺ in whole blood, serum, plasma and otherbody fluids obtained by the method of the present invention using an ionselective electrode. If the measured ionized Mg²⁺ in blood, serum orplasma is 0.5 mmol/l or less, then a bioavailable pharmaceuticallyacceptable magnesium salt which will result in a calculated dose of400-600 mg/day of elemental magnesium should be given. Calcium should beadministered in such a situation in the form of a bioavailablepharmaceutically acceptable calcium salt which will maintain the blood,plasma or serum level of ionized Ca²⁺ /ionized Mg²⁺ in the molar ratioof about 2:1 after the ionized Mg²⁺ level has been restored to thenormal optimal level of about 0.58-0.60 mmol/l. If, however, themeasured level of ionized Mg²⁺ is between about 0.5-0.6 mmol/l, then abioavailable pharmaceutically acceptable magnesium salt which willresult in a calculated dose of 300 mg/day of element magnesium should beadministered. Calcium, in this situation, should be administered in theform a of a bioavailable pharmaceutically acceptable calcium salt whichwill maintain the blood, plasma or serum level of ionized Ca²⁺ /ionizedMg²⁺ in the molar ratio of about 2:1 after the ionized Mg.sup. 2+ levelhas been restored to the normal optimal level of about 0.58-0.60 mmol/l.For prevention of magnesium deficiencies and disease states whichrequire a greater need for magnesium intake, a bioavailablepharmaceutically acceptable magnesium salt and a calcium salt which willresult in calculated doses of approximately 200 mg of elementalmagnesium and approximately 640 mg of elemental calcium, respectively,should be administered each day. This results in a molar ratio ofcalcium/magnesium of approximately 2:1.

It is a further object of the present invention to provide a mineralcomposition for prevention or treatment of magnesium deficiencies,magnesium imbalances, and calcium/magnesium imbalances based on themeasured ionized level of Mg²⁺ and ionized Ca²⁺ in whole blood, serumplasma and other body fluids of a neonate, infant and child obtained bythe method of the present invention using an ion selective electrode.Bioavailable pharmaceutically acceptable magnesium salt is administeredto the neonate, infant and child in a concentration sufficient to attainor maintain the normal physiological ionized Mg²⁺ levels in the blood.Bioavailable pharmaceutically acceptable calcium salt is administered ina concentration sufficient to maintain the blood, plasma, or serum levelof ionized Ca²⁺ /ionized Mg²⁺ in the molar ratio of about 1.9:1 to about2.6:1, more preferably 2.3:1 to about 2.5:1, most preferably about 2.5:1after the ionized Mg²⁺ level has been restored to the normalphysiological level.

Bioavailable magnesium salts include conventional pharmaceuticallyacceptable organic and inorganic dietary supplement salts of magnesiumsuch as magnesium oxide, magnesium phosphate, magnesium diphosphate,magnesium carbonate, magnesium aspartate, magnesium aspartatehydrochloride, magnesium chloride and the hydrates thereof, and thelike. Bioavailable calcium salts include conventional pharmaceuticallyacceptable organic and inorganic dietary supplement salts of calciumsuch as dibasic calcium phosphate or the like.

One embodiment of the present invention relates to a solid oral doseform composition. The composition may be in the form of conventionalpharmaceutical solid unit dosage forms such as a tablet, capsule, orsachet or the like, containing the magnesium and calcium components inthe requisite ratio.

The bioavailable magnesium may be in a controlled release form wherein,upon ingestion, the magnesium is released into the gastrointestinaltract over a prolonged period of time or in an uncontrolled instantrelease form, or combination thereof.

In one embodiment, the bioavailable magnesium salt is released from theformulation at an average percent rate at least equal to the averagepercent rate of release of the calcium salt.

Preparation of the composition into a solid oral dose form along withpharmaceutically acceptable carriers and excipient are described in U.S.Pat. No. 4,954,349.

The present mineral composition may be given alone, as a dietarysupplement, or may be administered with other minerals and/or withvitamins. One such multimineral dietary daily supplement includes, butis not limited to the following:

    ______________________________________                                                                Approximate Ele-                                                              mental                                                Mineral                 Concentration                                         ______________________________________                                        Calcium (as Dibasic Calcium Phosphate)                                                                320-1280  mg                                          Magnesium (as Magnesium Sulfate)                                                                      100-400   mg                                          Phosphorus (as Dibasic Calcium phosphate)                                                             125       mg                                          Iodine (as Potassium Iodide)                                                                          150       ug                                          Iron (as Ferrous Fumarate)                                                                            18        mg                                          Copper (as Cupric Oxide)                                                                              2         mg                                          Zinc (as Zinc Oxide)    15        mg                                          Manganese (as Manganese Sulfate)                                                                      2.5       mg                                          Potassium (as Potassium Chloride)                                                                     40        mg                                          Chloride (as Potassium Chloride)                                                                      36.3      mg                                          Chromium (as Chromium Chloride)                                                                       25        ug                                          Molybdenum (as Sodium Molybdate)                                                                      25        ug                                          Selenium (as Sodium Selenite)                                                                         25        ug                                          Vitamin K (as Phytonadione)                                                                           25        ug                                          Nickel (as Nickelous Sulfate)                                                                         5         ug                                          Tin (as Stannous Chloride)                                                                            10        ug                                          Silicon (as Sodium Metasilicate)                                                                      10        ug                                          Vanadium (as Sodium Metavanadate)                                                                     10        ug                                          ______________________________________                                    

The mineral composition of the present invention may also beadministered with one or more of the following vitamins:

    ______________________________________                                        Vitamin                  Concentrations                                       ______________________________________                                        Vitamin A (as Acetate and Beta Carotene)                                                               5000    I.U..sup.1                                   Vitamin E (as dl-Alpha Tocopheryl Acetate)                                                             30      I.U.                                         Vitamin C (Ascorbic Acid)                                                                              60      mg                                           Folic Acid               0.4                                                  Vitamin B-1 (as Thiamine Mononitrate)                                                                  1.5     mg                                           Vitamin B-2 (Riboflavin) 1.7     mg                                           Niacin (Niacinamide)     20      mg                                           Vitamin B-6 (as Pyridoxine HCl)                                                                        2       mg                                           Vitamin B-12 (Cyanocobalamin)                                                                          6       ug                                           Vitamin D (Calciferol)   400     I.U.                                         Biotin                   30      ug                                           Pantothenic Acid (as Calcium Pantothenate)                                                             10      mg                                           ______________________________________                                         .sup.1 I.U. = International Unit                                         

A further aspect of the present invention is a Ca²⁺ /Mg²⁺ mineralcomposition for use in an infant formula. The mineral compositionprovides a concentration of calcium and magnesium to ensure a ionizedmolar ratio of approximately 1.9 to about 2.6:1, more preferably 2.3:1to about 2.5:1 of ionized Ca²⁺ : ionized Mg²⁺.

An infant formula suitable for feeding neonates and infants comprisesprotein, carbohydrate, water, vitamins, minerals and an edible fat. TheCa²⁺ /Mg²⁺ mineral composition for the infant formula contains betweenabout 0.25 mmoles/kg/day to about 0.625 mmoles/kg/day of calcium in theform of a bioavailable pharmaceutically acceptable salt thereof, betweenabout 0.1 mmoles/kg/day to about 0.25 mmoles/kg/day of magnesium in theform of a bioavailable pharmaceutically acceptable salt thereof, inorder to achieve or maintain a molar ratio of ionized Ca²⁺ /ionized Mg²⁺in the blood of about 1.9 to about 2.6:1; more preferably 2.3:1 to about2.5:1, most preferably about 2.5:1.

The other components in the infant formula and concentrations areprovided in U.S. Pat. No. 4,670,285, and in: Textbook ofGastroenterology and Nutrition in infancy (2nd Ed) E. Lebenthal (Ed.)1989, Raven Press, NY, N.Y. pp. 435-458 incorporated herein byreference.

It is understood that the examples and embodiments described herein arefor illustrative purposes only and that various modifications or changesin light thereof will be suggested to persons skilled in the art and areto be included within the spirit and purview of this application andscope of the appended claims.

We claim:
 1. A composition for prevention or treatment of magnesiumdeficiencies or magnesium imbalances in an individual, the compositioncomprising as an active ingredient bioavailable magnesium salt, whereina concentration of bioavailable magnesium salt in said composition is anamount sufficient to maintain normal concentrations of ionized magnesiumin a range of approximately 0.53 to 0.67 mM for the biological sample ofwhole blood, serum or plasma in the individual.
 2. A composition forprevention or treatment of magnesium deficiencies or imbalances orcalcium deficiencies or imbalances, or calcium/magnesium imbalances inan individual, the composition comprising as the active ingredientsthereof,(A) bioavailable calcium salt; (B) bioavailable magnesium salt;and (C) wherein a concentration of the bioavailable calcium salt and thebioavailable magnesium salt in said composition is that amountsufficient to provide a normal molar ratio of ionized Ca²⁺ :ionized Mg²⁺in an individual wherein the normal molar ratio of ionized Ca²⁺ :ionizedMg²⁺ is selected from the group consisting of about 1:1 to about 2.5:1in whole blood, plasma or serum of an adult, about 1.9:1 to about 2.6:1in whole blood, plasma or serum of a neonate, infant or child and about0.90:1 to about 1.15:1 in cerebral spinal fluid.
 3. A composition ofclaim 2 wherein the normal molar ratio of ionized Ca²⁺ :ionized Mg²⁺ inwhole blood, plasma or serum of an adult is about 2:1.
 4. A compositionof claim 2 wherein the concentration of calcium salt is about 640 mg. 5.A composition of claim 2 wherein the concentration of magnesium salt isabout 200 mg.
 6. A dietary supplement comprising the composition ofclaim 1 or
 2. 7. An infant formula useful in providing normal ionizedCa²⁺ and normal ionized Mg²⁺ concentrations in a neonate or infant, saidformula comprising conventional ingredients of the formula and thecomposition of claim 1 or
 2. 8. The infant formula of claim 7 whereinthe conventional ingredients comprises protein, carbohydrate, water,vitamins, minerals and an edible fat.
 9. A composition of claim 2wherein the concentration of calcium salt is about 320 mg to about 1280mg.
 10. A composition of claim 2 wherein the concentration of magnesiumsalt is about 100 mg to about 400 mg.
 11. A composition for preventionor treatment of magnesium deficiencies or magnesium imbalances in anindividual, the composition comprising as an active ingredientbioavailable magnesium salt, wherein a concentration of bioavailablemagnesium salt in said composition is an amount sufficient to maintainnormal concentrations of ionized magnesium in a range of approximately1.10-1.23 mM for the biological sample of cerebral spinal fluid in theindividual.